Treatment of cellular proliferative disorders with compositions comprising Hom polypeptide (also known as Xom, VENTX2)

ABSTRACT

Disclosed are compositions comprising Hom polypeptide (also known as Xom, Ventx2) and methods for treating cellular proliferative disorders comprising. Also disclosed are diagnosis methods, prognosis methods, and drug screening methods.

RELATED APPLICATION

This application claims priority of U.S. Provisional Application No.60/775,645, filed Feb. 22, 2006. The prior application is incorporatedherein by reference in its entirety.

BACKGROUND

One of the fundamental questions facing development biologist is how amulti-cell organism develops from a fertilized egg, a totipotent stemcell, which contains a simple symmetrical structure, into an adult,which has a three-dimensional body plan. Dysregulation of this processresults in aborted embryogenesis during early development and frequentlyresults in tumor formation in adult life. There is a need for agents andmethods for regulating the development process and treating variousdisorders related to dysregulated development, such as cancers,degenerative diseases, and immune disorders.

The Wnt signaling pathway is involved in the proliferation ordifferentiation of various stem cells. For example, it plays essentialroles in the differentiation of hematopoietic stem cells derived fromfetal tissue or bone marrow. Bone morphogenic proteins (BMPs) belong tothe TGF-β super family and are found in species ranging from flies tomammals. The BMP signal pathway is important in cell fate determinationand pattern formation during embryogenesis and in the maintenance oftissue homeostasis in the adult. The BMP pathway is also involved inregulation of morphogenesis and postnatal regeneration of GIdevelopment. See, e.g., U.S. Pat. Nos. 6,824,971, 6,159,462, 6,465,249,and 6,165,748.

SUMMARY

This invention relates to treating cancers, degenerative diseases, andimmune disorders, and identifying compounds for treating these disordersusing the Wnt/Bβ-catenin or BMP4/Xom signaling pathway. Shown below arethe polypeptide and nucleotide sequences of Xom and its human homologueHom.

Xom polypeptide (SEQ ID NO: 7):mtkafssvew laqssrrshr eqpskvdqry spypspslpswnsdvspssw nsqlspdpds aqvspcpasa qvspyssdseislysheeea sfygmdlnts sspgdngllh semvsvpdniprassdedaa ksayststds gyesetscss stapegdaislspndtsdee gkmgrrlrta ftsdqistle ktfqkhrylgaserqklaak lqlsevqikt wfqnrrmkyk reiqdgrpdsyhpaqffgvy gyaqqptpvf qhavqhpypg ynplmetlpgtmpytmhppa mdsmtpfnsq pfqmlylpqq hlgqpltyqe erpfvry(Underlined: Ser140 and Ser144 residues, andaa.176-233/homeodomain/SEQ ID NO.: 3)Xom cDNA nucleotide sequence (SEQ ID NO: 10):agaacacaag gactaataca gacaagatga ctaaagctttctcctctgtt gaatggcttg ctcaaagcag ccgcagatctcacagagagc agccaagcaa agtggatcag agatattcaccgtaccccag gccatccctg ccttcctgga acagtgatgtgtccccttct tcatggaaca gccaactatc tccagatccagacagtgccc aagtctcacc atgccctgtg agtgcacaagtatctccata ttcctcagac agtgaaatat cactgtattcacatgaagaa gaagcctctt tctatggaat ggactttaatacatcatcat cccctggaga caatggattg ctacacagggacacaacctc atactccaga ggaatggagg ccatgtcggccagcactcca gcaacatcac ctgtgaaagg ggcacaacctgttgattccg cctacagcac tagcactgac tcaggctatgaaagtgaaac gagtcgatcc aactctacag cccctgaaggagatgcctcc gtatctctga gtcccaatga tacctcagatgaagagggca agatgggccg aaggttgagg acggctttcaccagtgatca gatctccact ctggagaaga cttttcagaaacacagatac cttggggcgt ctgaaagacg gaaactcgcagccaaactcc agctttctga agtccagatt aaaacttggttccagaaccg caggatgaaa tacaaacggg aaatccaagatggcagacca gactcatacc acccagccca gttctttggtgtgtacggct atgcacagca gcccactcct gtattccagcatgcagtcca acatccctac ccaggttata acccactaatggaaaccctg cctggtacca tgccctatac catgcatccacctgccatgg actctctgac tcccttcaac tctcaaccttttcagatgct ctacctgccc caacagcacc ttgggcaacctctggcctat taggaagaaa ggccatttgt tagatattaatctagaactt ataaaaggac tatactaaag gctggacttttccatggact tctgtcctcc cgcaggacaa acaaaattgcactgaatatt gttattgaca agatgtttac tgaatggatggctaatattg ggccatgtgt tgacatgatt ttattcacattgaatagtgg cgtgtatatt ctatgaaaaa taccatttatatgactaata aatgtaagtt atatttaaaa aaaaaaaaaa a(underlined: (nt 21 to 1013)/SEQ ID NO: 4;coding sequence (nt27 to 1013)/SEQ ID NO: 8)Hom polypeptide (SEQ ID NO: 5):mrlssspprg pqqlssfgsv dwlsqsscsg pthtprpadfslgslpgpgq tsgareppqa vsikeaagss nlpapertmaglskepntlr aprvrtaftm eqvrtlegvf qhhgylsplerkrlaremql sevqiktwfq nrrmkhkrqm qdpqlhspfsgslhappafy stssglangl qllcpwapls gpqalmlppgsfwglcqvaq ealasagasc cgqplashpp tpgrpslgpa lstgprglca mpqtgdaf(Underlined: aa, 93-151/homeodomain/SEQ ID NO.: 1)Hom nucleotide sequence (SEQ ID NO: 9):acctggccgc catgcgcctc tcctcctccc cacctcgtggcccgcagcag ctctccagct ttggctccgt ggactggctctcccagagca gctgctcagg gccgacccac acccccaggcctgccgactt ctccctgggg agcctccctg gcccaggccagacatccggc gcccgggagc cccctcaggc cgtcagcatcaaggaggccg ccgggtcctc aaatctgcct gcgccggagaggaccatggc cgggttgagt aaggagccaa ataccttgcgggccccccgt gtccgcacag ccttcaccat ggagcaggtccgcaccttgg agggcgtctt ccagcaccac cagtacctgagccctctgga gcggaagagg ctggccaggg agatgcagctctcagaggtc cagataaaaa cctggtttca gaatcgccgcatgaaacaca aacggcaaat gcaggacccc cagctgcacagccccttctc ggggtctctc catgcgcccc cagctttctactcaacgtct tctggccttg ccaatggcct gcagctgctgtgcccttggg cacccctgtc cgggccccag gctctgatgctgccccctgg ctccttctgg ggtctctgcc aagtggcacaagaggccctg gcatctgcgg gagcttcctg ctgcgggcagcctctggcgt cccacccccc taccccaggc cggccttcgctgggaccagc cctgtccacg gggccccggg gcctgtgtgctatgccacag acgggggatg cattttgagg aggcacctctgactcccaca ctcgcggtct tgctgatcgc acctggctcctacctggagg actcagttgt tctgtttaca tcctggtggcacctctcacc ctgacccaca caaaggttct ggagattactggagaatata tataaatata tatatgtacg tatatatgtaaatacacata tacgtatata taaatatata tatacatatgtgtgtgtata tatatatata tttttttttt tttttttttttttgagacgg agtgttgctc tgtcacccag gctggagtgcaatgacgcaa tctcggctca ctgcaacctc cgcctcctgggttcaagcga ttctccagcc tcagcctccc gagtagctgggattacagac acccgccacc acgcccggct aattttttctatttttagta gaaatggggt ttcaccatgt tagccaggctggtctcaaac tcctgaccct gtgatccgcc cgcctcggcctcccaaagtg ctgggattac aggcatgagc cactgcacccggccctgaga atatatttat taaagccacc tcttcactgaaagttaccga aagagtcggt ttaggaagga aacgaagggtcagtgaacag agtcaaatgc agaagtgggc ttgtcatgggtagggctttc ggcgtacgat aaaaggatca tttgttttttaaaaggggtt ggaaaaactg gttttccagt tggaaacagtaaaggttgta agctttgtgt gtacaaaaga aaacagggaatgcaggtgtg tttatagcgt tgtggttcaa gtccctcttaacaagaactc caaagctgga aagcaggagg gaacaaaggtgaacatgaag gcgaggatgc tggggccctg cagtgcgctctaggctgtgc gtgagccggg actgtaccca cagcttgctgagggctgctc ttcttgggcc agggaaagca gggcagccgggacctgcggc tgtgcctgga ctgaagctgt cccgcaggtccccaccctcc aacacgtgct cacctgtccc cctcctcgcagcagcctcgg gacaaaacaa tgactcaagg acagcacttctcgcagaagg tctggaagtg cccagaatgg gaggcacggaagcccctccc ggggaggact cccgcgttga tggaccgttcttggtgcaga ctcctgactg cgtgcatgaa acctgagacaagtgcaattc cttccatgtc gccccagagt gcccaggaggcaggcagtgc ggggtgccca ggcagacggg ttcagcctgcagaactggag gcgacctgtg aaacccaccc gggcaccccaacaggaacag aagcgtggtc ctgcggctgc gtccccagcgagtttcactt tccccttgct cgtttctccc ttgttgtaagtgtttacaac tggcatgtgc ttttaaacgt caggtaagaggggaacagct gctgtacatc gtcctggcga gtgacaatgtgacagaagcc tgggcgaggc cctcggaggg cagcagctggacaggggcta ctgggtttgg cctggacagc actgatttgtggatgtggat gggggcacgt tgtccgtgat aaaagtacaagtgcccctca caaaaaaaaa aaaaaaaa(Underlined: coding sequence (nt12 to 788/ SEQ ID NO: 6)

In one aspect, the invention features a method for treating a cellularproliferative disorder in a subject. A cellular proliferative disorderrefers to a disorder characterized by uncontrolled, autonomous cellgrowth, including malignant and non-malignant growth. The methodincludes administering to a subject in need thereof an effective amountof a polypeptide containing SEQ ID NO: 1 or 3, or a functionalequivalent thereof. A “functional equivalent” refers to a polypeptidederivative of a common polypeptide, e.g., a protein having one or morepoint mutations, insertions, deletions, truncations, a fusion protein,or a combination thereof, and retaining substantially the ability of thecommon polypeptide, such as binding to a LEF1/TCF. In one example, thepolypeptide lacks and LEF1/TCF transactivation domain. The cellularproliferative disorder can be a condition characterized by aberrantactivation of LEF1/TCF-mediated transcription. An aberrant activation ofLEF1/TCF-mediated transcription refers to a cellular condition where theLEF1/TCF-mediated transcription is abnormally high, as determined by theTOPflash assay described in Example 1 below or any analogous assays.

The invention also features a method for treating aninflammation-related disorder in a subject. An inflammation-relateddisorder is characterized by a local or systemic, acute or chronicinflammation. The method includes administering to a subject in needthereof an effective amount of an inhibitor of a polypeptide containingSEQ ID NO: 1 or 3 (e.g., Xom or Hom) or a functional equivalent thereof.The inflammation-related disorder is an auto-immune disorder or aninflammatory disorder. An inhibitor of a polypeptide containing SEQ IDNO: 1 or 3 refers to a compound that reduces the protein level in a cellin a statistically significant manner. Examples of the inhibitor includean antibody, an antisense nucleic acid, and an RNAi agent, as well assmall molecule compounds and naturally occurring compounds, which targetHom/Xom.

The invention also features a method for treating a degenerativedisorder in a subject. A degenerative disorder is characterized by alocal or systemic, acute or chronic degeneration, loss of cellularvolume or cellular function. The method includes administering to asubject in need thereof an effective amount of an inhibitor of apolypeptide containing SEQ ID NO: 1 or 3 (e.g., Xom or Hom) or afunctional equivalent thereof.

The invention further features a method for treating myelodysplasticsyndromes. The method includes administering to a subject in needthereof an effective amount of an inhibitor of a polypeptide containingSEQ ID NO: 1 or 3, or a functional equivalent thereof.

In another aspect, the invention features a screening method ofidentifying a compound for treating a cellular proliferative disorder.The method includes contacting a test compound with a polypeptidecontaining a fragment of SEQ ID NO: 3 that include Ser 140 or Ser 144;and determining the phosphorylation of Ser 140 or Ser 144 (e.g., by aSer 140 and Ser 144 phospho-specific antibody). The phosphorylationlevel in the presence of the compound, if lower than that in the absenceof the compound, indicates that the compound is a candidate for treatingthe disorder. The screening method can also be conducted by contacting atest compound with a cell having a polypeptide containing a fragment ofSEQ ID NO: 3 that includes Ser 140 or Ser 144; and determining thephosphorylation of Ser 140 or Ser 144. The phosphorylation level in thepresence of the compound, if lower than that in the absence of thecompound, indicates that the compound is a candidate for treating thedisorder. The fragment is at least 14 (e.g., 15, 18, 20, 30, 50, 100,150, 200, 250, and 300) amino acid residues in length.

The above method can also be used to identify compounds that increasethe phosphorylation of Ser 140 or Ser 144. Compounds thus-identifiedrepresent candidates for treating for treating cellular degenerativedisorders of inflammation-related disorders. Specifically, if thephosphorylation is higher than control, it indicates that the compoundis a candidate for treating cellular degenerative disorder.

In yet another aspect, the invention features a composition thatcontains a polypeptide having the sequence of SEQ ID NO: 1 or 3, or afunctional equivalent thereof, or an activator of the polypeptide. Anactivator of the polypeptide is a compound that increases the proteinlevel of the polypeptide by either inducing its expression or repressingits proteolysis. Examples of the activator include retinoic acid,resveratrol, ellagic acid, aspirin, and their derivatives. Thecomposition can further include salicylic acid, emodin, flavonoid, ortheir derivatives. In one embodiment, the composition is a topicalcomposition, which can be used for skincare. Specifically, one canadminister to a subject in need thereof a safe and effective amount ofthe composition. In another embodiment, the composition is a dietarycomposition, such as a tea, soft drink, juice, milk, coffee, jelly, icecream, yogurt, cookie, cereal, chocolate, snack bar, candy, chewing gum,syrup, or food capsule. This dietary composition can be used to treat orslow down the onset of a cellular proliferative disorder.

In a further aspect the invention features a method of assessing asubject's cancer prognosis. The method includes obtaining a biologicalsample from the subject; and determining the presence of a gene encodinga polypeptide containing SEQ ID NO: 1 in the sample. The subject isdetermined to have a good prognosis if the gene is present on bothchromosomes or to have a bad prognosis if the gene missing from one orboth of the chromosomes.

One can also assess a subject's cancer prognosis by obtaining abiological sample from the subject; and determining the expression levelof a gene encoding a polypeptide containing SEQ ID NO: 1 in the sample.The subject is determined to have a good prognosis if the expressionlevel is above a control level or to have a bad prognosis if theexpression level is below the control level. The control level can beobtained from a normal subject. The method can further comprisecontacting the sample with a chemotherapeutical agent prior todetermining the express level of a gene encoding a polypeptidecontaining SEQ ID NO: 1 in the sample. The biological sample can be atumor biopsy sample or a blood sample.

Also featured is a method for diagnosing of myelodysplastic syndromes.The method includes obtaining a biological sample from the subject; anddetermining the expression level of a gene encoding a polypeptidecontaining SEQ ID NO: 1, e.g., Hom, in the sample. An abnormal increasedexpression level indicates the subject has or is prone to developmyelodysplastic syndromes.

Within the scope of the invention is a method for determining whether asubject has or is prone to develop systemic lupus erythematosus (SLE).The method includes obtaining a biological sample from the subject; anddetermining the expression level of a gene encoding a polypeptidecontaining SEQ ID NO: 1 in the sample. The subject is determined to haveor be prone to develop SLE syndromes if the expression level is above acontrol level.

Within the scope of this invention is a method for maintaining apluripotent cell, the method comprises contacting the cell with anactivator of a polypeptide containing SEQ ID NO: 1. The pluripotent cellcan be a stem cell, such as a hematopoietic stem cell, agastrointestinal stem cell, a neuronal stem cell, and a skin stem cell.

Also within the scope of this invention are isolated mutant polypeptidesof Xom or Horn that include the sequence of SEQ ID NO: 1 or 3. Examplesof such mutant polypeptides include: XomND55, Xom ND175, XomCD145, andXomCD85, which corresponds to aa. 56-326, aa. 176-326, aa. 1-181, andaa. 1-241 of SEQ ID NO: 7 (SEQ ID NOs: 11-14, respectively). Otherexamples include the fusion of aa 1-130 and aa 241-326 of SEQ ID NO: 7(SEQ ID NO: 15) and the fusion of Xom aa1-175 and plus Horn aa 93-258(SEQ ID NO: 16). Other examples include a polypeptide containing afragment of SEQ ID NO: 7 that includes Ser 140 or Ser 144, such asTDSGYESETSC (SEQ ID NO: 2) and its mutants in which Ser 140 or Ser 144is substituted by other amino acid residues such as alanine. Thesepolypeptides are least 11 (e.g., 11, 13, 15, 20, 50, 100, 150, 200, 250,and 300) amino acid residues in length. They can be used to screeninginhibitors of Ser 140 or Ser 144 phosphrylation or as therapeutic agentsto inhibit Ser 140 or Ser 144 phosphrylation of endogenous Xom. Withinthe scope of this invention are fusion proteins containing one or moreof the afore-mentioned mutant sequences and a heterologous sequence. Aheterologous polypeptide, nucleic acid, or gene is one that originatesfrom a foreign species, or, if from the same species, is substantiallymodified from its original form. Two fused domains or sequences areheterologous to each other if they are not adjacent to each other in anaturally occurring protein or nucleic acid.

An isolated polypeptide refers to a polypeptide free from naturallyassociated molecules, i.e., its is at least 75% (i.e., any numberbetween 75% and 100%, inclusive) pure by dry weight. Purity can bemeasured by any appropriate standard method, for example, by columnchromatography, polyacrylamide gel electrophoresis, or HPLC analysis. Anisolated polypeptide of the invention can be purified from a naturalsource, produced by recombinant DNA techniques.

The invention also features an isolated nucleic acid that contains asequence encoding the just-mentioned mutant or fusion polypeptide or acomplement of the sequence. A nucleic acid refers to a DNA molecule(e.g., a cDNA or genomic DNA), an RNA molecule (e.g., an mRNA), or a DNAor RNA analog. A DNA or RNA analog can be synthesized from nucleotideanalogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA. An “isolatednucleic acid” is a nucleic acid the structure of which is not identicalto that of any naturally occurring nucleic acid or to that of anyfragment of a naturally occurring genomic nucleic acid. The termtherefore covers, for example, (a) a DNA which has the sequence of partof a naturally occurring genomic DNA molecule but is not flanked by bothof the coding sequences that flank that part of the molecule in thegenome of the organism in which it naturally occurs; (b) a nucleic acidincorporated into a vector or into the genomic DNA of a prokaryote oreukaryote in a manner such that the resulting molecule is not identicalto any naturally occurring vector or genomic DNA; (c) a separatemolecule such as a cDNA, a genomic fragment, a fragment produced bypolymerase chain reaction (PCR), or a restriction fragment; and (d) arecombinant nucleotide sequence that is part of a hybrid gene, i.e., agene encoding a fusion protein. The nucleic acid described above can beused to express the polypeptides of this invention. For this purpose,one can operatively link the nucleic acid to suitable regulatorysequences to generate an expression vector.

A vector refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. The vector can becapable of autonomous replication or integrate into a host DNA. Examplesof the vector include a plasmid, cosmid, or viral vector. A vector ofthis invention includes a nucleic acid in a form suitable for expressionof the nucleic acid in a host cell. Preferably the vector includes oneor more regulatory sequence operatively linked to the nucleic acidsequence to be expressed. a “regulatory sequence” includes promoters,enhancers, and other expression control elements (e.g., polyadenylationsignals). Regulatory sequences include those that direct constitutiveexpression of a nucleotide sequence, as well as tissue-specificregulatory and/or inducible sequences. The design of the expressionvector can depend on such factors as the choice of the host cell to betransformed, the level of expression of protein desired, and the like.the expression vector can be introduced into host cells to produce thepolypeptide of this invention. Also within the scope of this inventionis a host cell that contains the above-described nucleic acid. Examplesinclude E. coli cells insect cells (e.g., using baculovirus expressionvectors), yeast cells, or mammalian cells. See e.g., Goeddel, (1990)Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif.

To produce a mutant or fusion polypeptide of this invention, one canculture a host cell in a medium under conditions permitting expressionof the polypeptide encoded by a nucleic acid of this invention, purifythe polypeptide from the cultured cell or the medium of the cell.Alternatively, the nucleic acid of this invention can be transcribed andtranslated in vitro, for example, using T7 promoter regulatory sequencesand T7 polymerase.

The details of one or more embodiments of the invention are set forth inthe description below. Other features, objects, and advantages of theinvention will be apparent from the description and from the claims.

DETAILED DESCRIPTION

This invention is based, at least in part, on the unexpected discoveriesof a signal transduction component that functions as a point ofconvergence to mediate the combined signaling of the Wnt/Bβ-catenin andBMP4/XOM pathways.

Xom (also known as Vent2, Vox, and Xbr-1), is a cell fate determinationfactor of the Vent family of Homeobox genes. It is both atranscriptional repressor and an activator (Ladher et al. 1996,Development 122, 2385-2394; Onichtchouk et al., 1996 Development 122,3045-3053; Schmidt et al., 1996, Development 122, 1711-1721; andPapalopulu et al., 1996, Dev Biol 174, 104-114). During earlyembryogenesis, Xom is implicated in the formation of ventral mesodermand in defining the dorsoventral patterning (Onichtchouk et al., 1998,Development 125, 1447-1456 and Koide et al., 2005. Proc Natl Acad SciUSA 102, 4943-4948). Zygotic Xom Transcription starts after midblastulatransition (MBT) and distributes from a more ubiquitous expressionpattern during the early gastrula stage to the ventrol-lateral regionsas gastrulation proceeds (Ladher et al. 1996, Development 122, 2385-2394and Schmidt et al., 1996, Development 122, 1711-1721). The expression ofXom appears to be positively regulated by signals from the ventralsignal center, such as the BMP4, but negatively regulated bydorsal-specific genes, such as the Gooscoid (Gsc) and the noggin (Ladheret al. 1996, Development 122, 2385-2394; Onichtchouk et al., 1996,development 122, 3045-3053). Xom expression in turn contributes to theformation of the dorsoventral pattern by promoting the expression ofventral genes such as the BMP4 and the Vent genes and inhibiting theexpression of dorsal-organizer genes such as the Gsc and chordin(Onichtchouk et al., 1996, Development 122, 3045-3053; and Schmidt etal., 1996, Development 122, 1711-1721). To exert its transcriptionalrepressor function, Xom binds directly to the distal element of thedorsal specific gene promoters, such as the Gsc, and inhibit theirtranscription (Trindade et al., 1999, Dev Biol 216, 442-456).

As described herein, Xom interacts functionally with the LEF1/TCFstranscription factors. The LEF1/TCFs are a family of high mobility group(HMG) transcriptional factors that possess no intrinsic transcriptionalactivities. Rather, the LEF1/TCF-mediated transcription activities aretightly controlled by their associated factors (Hurlstone et al., 2002,Embo J 21, 2303-2311). In a non-induction state, the LEF1/TCFs areassociated with transcriptional repressors, such as the Grouch, andCtBP, which maintain the LEF1/TCF-mediated transcription in a repressedstate (Roose et al., 1998, Nature 395, 608-612; Brantjes et al., 2001,Nucleic Acids Res 29, 1410-1419; Cavallo et al., 1998, Nature 395,604-608; Waltzer et al., Nature 395, 521-525; and Brannon et al., 1999,Development 126, 3159-3170). During early embryogenesis, localenrichment of β-catenin in the future dorsal side of embryos allows itto interact with LEF1/TCFs and to induce the expression ofdorsal-specific genes, such as Siamois, Twin, and Xnr (Harland et al.,1997, Annu Rev Cell Dev Biol 13, 611-667; Brannon et al., 1997, GenesDev 11, 2359-2370; Laurent et al., 1997, Development 124, 4905-4916; andMcKendry et al., 1997, Dev Biol 192, 420-431. Besides determination ofcell fate during early embryogenesis, excessive activation ofLEF1/TCF-mediated transcription by β-catenin has also been implicated asthe initial step of malignant transformation of a variety of cancers(Barker et al., 2000, Adv Cancer Res 77, 1-24). The LEF1/TCF-factors arethe transcriptional mediators of Wnt/β-catenin, therefore, the LEF1/TCFpromoter-luciferase reporter activity has generally been regarded as anindicator of Wnt/β-catenin activities.

The role of LEF1/TCFs in ventral cell fate determination is less clear,although several studies indicate their potential involvement in theprocess. For example, expression profiling showed that members ofLEF1/TCF family are broadly distributed in ventral-posterior regions(Molenaar et al., 1998, Mech Dev 75, 151-154 and Oosterwegel et al.,1993, Development 118, 439-448). Mutagenesis studies revealed thatLEF1−/−TCF1−/− mice carry caudal defects with neural expansion (Galceranet al., 1999, Genes Dev 13, 709-717) and that loss of function of LEF1leads to ventral rather than dorsal defects in Xenopus (Roel et al.,2002, Curr Biol 12, 1941-1945). Consistent with the possible involvementof LEF1/TCFs in ventral cell fate determination, promoter analysisrevealed that many ventral genes, such as Xom and Bambi, containLEF1/TCF binding sites. Mutations of the LEF1/TCF binding site of theseventral genes cause significant inhibition of their responsiveness tothe BMP4 signaling (Karaulanov et al., 2004, Embo J 23, 844-856).

As described herein, it was found that Xom and LEF1/TCF-factorsfunctionally interact with each other. This interaction plays anessential role in stem cell pool maintenance and cell fate determinationduring early embryogenesis and serves as a point of convergence tomediate the combined signaling effect of BMP/Xom and Wnt/β-cateninpathways during early embryogenesis. A human homologue of Xom, Hom, wascloned. It was found to function in a manner similar to that of XenopusXom. In particular, it was found that over expression of Xom/Hom incancer cells or terminal differentiated cells results in cell growtharrest or cell death.

Also, when combined together, expression of the LEF1/TCF factors and Xomor forced expression of Hom induces cell death in colon cancer cells,with the efficacy of near 100%. Similar effects were found in cervicalcancer cells and prostate cancer cells. Further, it was found that anumber of cancer chemotherapy drugs or treatment, e.g., 5-FU, DOX, andradiations, induced the expression of Xom or Hom. These discoveriessuggest that Xom/Hom behaves like a tumor suppressor and can be used incancer diagnosis, prognosis, and treatment. The fact that the geneencoding Hom is located at 10q26, which is prone to loss duringoncogenesis and cancer metastasis, supports the roles of Hom/Xom incancer diagnosis, prognosis, and treatment.

Hom/Xom's stability is critical for stem cell pool maintenance andcell-fate determination. The protein level of Xom is controlled byproteolysis, which in turn is controlled by phosphorylation ofSer140/144. During development, endogenous Xom was rapidly degraded atthe onset of gastrulation. Xom Ser140/144 was not phosphorylated duringthe pre-gastrulation period but become rapidly phosphorylated at theonset of gastrulation, a pattern in reciprocal relationship to the XomStability. It was also found that the phospho-moiety plays a criticalrole in mediating the binding between Xom and β-TRCP (the E3 ligase thatmediates Xom protreolysis) and that non-phosphorylatable Xom mutant isresistant to proteolysis. Furthermore, while expression of wild-type Xomalone causes growth arrest in 30% colon cancer cells, expression ofstable Xom mutant causes growth arrest in 60% colon cancer cells. Thisphosphorylation and proteolysis processes represent novel therapeutictargets for identifying new drugs for treating cancer and otherdisorders.

Besides the stability, the expression of Hom/Xom is also crucial ofvarious development processes. For example, it was found that Homproduction can be induced by GM-CSF and IL4 in the CD14+ bloodprogenitor cells. This induction leads to terminal differentiation ofthe CD14+ cells (which are a monocyte progenitor cell) into dendriticcells to present antigen and thereafter undergo apoptosis.

Dendritic cells are immune cells and form part of the mammalian immunesystem. Their main function is to process antigen material and presentit on their surface to other cells of the immune system. Dendritic cellsare present in small quantities in tissues that are in contact with theexternal environment, mainly the skin (where they are often calledLangerhans cells) and the inner lining of the nose, lungs, stomach andintestines. They can also be found at an immature state in the blood.Once activated, they migrate to the lymphoid tissues where they interactwith T cells and B cells to initiate and shape the immune response.Altered function of dendritic cells plays a major a key role in allergyand autoimmune diseases like lupus erythematosus. Allergy is apathologically overblown reaction to an outside allergen; autoimmunediseases are erroneous immune reactions to the organism's own antigen.See, e.g., Santiago-Schwarz et al. J. Immunol. 2001 Aug. 1;167(3):1758-68. Given the roles of dendritic cells, modulatingexpression of Xom/Hom in dendritic cells presents a way to treatautoimmune disease and inflammation. For example, Xom/Hom modulates thedifferentiation of dendritic cells in at least two stages, i.e. (i) anearlier stage when CD14+ blood progenitor cells, in response to GM-CSFand IL4, terminally differentiate into dendritic cells and (ii) aterminal stage when terminally differentiated dendritic cells havealready formed as, e.g., Langerhans cells in the skin. At the earlierstage, Xom/Hom expression or activity is required for CD14+ bloodprogenitor cells differentiation into progenitor of dendritic cells.Accordingly, blocking Xom/Hom expression or activity can lead to fewerdendritic cells, thereby reducing unwanted immune response. At theterminal stage, on the other hand, high level of Xom/Hom expression oractivity forces the terminally differentiated dendritic cells intoapoptosis. It follows, at this stage, high level of Xom/Hom expressionor activity depletes, via inducing apoptosis, the terminallydifferentiated dendritic cells, thereby also reducing unwanted immuneresponse, in particular, that in the skin.

Diagnostic and Prognostic Assays

A cancer cell or a cell prone to tumorigenesis can be detected in asubject based on the absence of the Hom polypeptide (e.g., antibody) ora nucleic acid (e.g., genomic DNA or mRNA) encoding the polypeptide in atest sample from the subject. In other words, the polypeptide andnucleic acids can be used as markers to indicate the presence or absenceof a cancer cell. Diagnostic and prognostic assays of the inventioninclude methods for assessing the expression level of the Hompolypeptide or nucleic acid and for identifying variations and mutationsin the sequence of the Hom polypeptide or nucleic acid.

The presence, level, or absence of the Hom polypeptide or nucleic acidin a test sample can be evaluated by obtaining a test sample from a testsubject and contacting the test sample with a compound or an agentcapable of detecting the Hom polypeptide or nucleic acid (e.g., mRNA orgenomic DNA probe). The “test sample” includes tissues, cells andbiological fluids isolated from a subject, as well as tissues, cells andfluids present within a subject. The level of expression of the Hom genecan be measured in a number of ways, including measuring the mRNAencoded by the Hom gene; measuring the amount of polypeptide encoded bythe Hom gene; or measuring the activity of polypeptide encoded by theHom gene.

The level of mRNA corresponding to the Hom gene in a cell can bedetermined both by in situ and by in vitro formats. Messenger RNAisolated from a test sample can be used in hybridization oramplification assays that include, Southern or Northern analyses, PCRanalyses, and probe arrays. One preferred diagnostic method for thedetection of mRNA levels involves contacting the isolated mRNA with anucleic acid probe that can hybridize to the mRNA encoded by the Homgene. The probe can be a full-length Hom nucleic acid, such as thenucleic acid of SEQ ID NO: 6 or a portion thereof, such as anoligonucleotide of at least 10 nucleotides in length and sufficient tospecifically hybridize under stringent conditions to Hom mRNA or genomicDNA.

In one format, mRNA (or cDNA prepared from it) is immobilized on asurface and contacted with the probes, for example, by running theisolated mRNA on an agarose gel and transferring the mRNA from the gelto a membrane, such as nitrocellulose. In another format, the probes areimmobilized on a surface and the mRNA (or cDNA) is contacted with theprobes, for example, in a gene chip array. A skilled artisan can adaptknown mRNA detection methods for use in detecting the level of mRNAencoded by the Hom gene.

The level of mRNA (or cDNA prepared from it) in a sample encoded by Homgene can be evaluated with nucleic acid amplification, e.g., by standardPCR (U.S. Pat. No. 4,683,202), RT-PCR (Bustin S. J. Mol Endocrinol.25:169-93, 2000), quantitative PCR (Ong Y. et al., Hematology. 7:59-67,2002), real time PCR (Ginzinger D. Exp Hematol. 30:503-12, 2002), and insitu PCR (Thaker V. Methods Mol Biol. 115:379-402, 1999), or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques known in the art. As used herein,amplification primers are defined as being a pair of nucleic acidmolecules that can anneal to 5′ or 3′ regions of a gene (plus and minusstrands, respectively, or vice-versa) and contain a short region inbetween. Under appropriate conditions and with appropriate reagents,such primers permit the amplification of a nucleic acid moleculecomprising the nucleotide sequence flanked by the primers.

For in situ methods, a cell or tissue sample can be prepared andimmobilized on a support, such as a glass slide, and then contacted witha probe that can hybridize to genomic DNA on chromosomes or mRNA thatencodes the Hom polypeptide.

In another embodiment, the methods of the invention further includecontacting a control sample with a compound or agent capable ofdetecting Hom mRNA, or genomic DNA, and comparing the presence of HommRNA or genomic DNA in the control sample with the presence of Hom mRNAor genomic DNA in the control sample with the presence of Hom mRNA orgenomic DNA in the test sample.

The above-described nucleic acid-based diagnostic methods can providequalitative and quantitative information to determine whether a subjecthas or is predisposed to a disease associated with aberrant Hom geneexpression, e.g., cancers.

A variety of methods can be used to determine the level of Hompolypeptide. In general, these methods include contacting an agent thatselectively binds to the polypeptide, such as an antibody, to evaluatethe level of polypeptide in a sample. Antibodies can be polyclonal, ormore preferably, monoclonal. An intact antibody, or a fragment thereof(e.g., Fab or F(ab′)₂) can also be used. In a preferred embodiment, theantibody bears a detectable label. The term “labeled”, with regard tothe probe or antibody, is intended to encompass direct labeling of theprobe or antibody by physically linking a detectable substance to theprobe or antibody, as well as indirect labeling of the probe or antibodyby reactivity with a detectable substance. For example, an antibody.with a rabbit Fe region can be indirectly labeled using a secondantibody directed against the rabbit Fe region, wherein the secondantibody is coupled to a detectable substance. Examples of detectablesubstances are provided herein. Appropriate detectable substance orlabels include radio isotopes (e.g., ¹²⁵I, ¹³¹I, ³⁵S, ³H, or ³²P),enzymes (e.g., alkaline phosphatase, horseradish peroxidase, luciferase,or β-galactosidase), fluorescent moieties or proteins (e.g.,fluorescein, rhodamine, phycoerythrin, GFP, or BFP), or luminescentmoieties (e.g., Qdot™ nanoparticles supplied by the Quantum DotCorporation, Palo Alto, Calif.).

The detection methods can be used to detect the Hom polypeptide in abiological sample in vitro as well as in vivo. In vitro techniques fordetection of the Hom polypeptide include ELISAs, immunoprecipitations,immunofluorescence, EIA, RIA, and Western blotting analysis. In vivotechniques for detection of the Hom polypeptide include introducing intoa subject a labeled anti-Hom antibody. For example, the antibody can belabeled with a detectable substance as described above. The presence andlocation of the detectable substance in a subject can be detected bystandard imaging techniques.

Xom Ser140/144 phosphorylation levels can also be used as an indicatorof tumorigenicity. More specifically, higher ser140/144 phosphorylationthan control (or kinase activity) indicates a higher likelihood ofdeveloping tumor/cancer. Ser140/144 phosphorylation can be measuredusing the Xom related sequence and Ser140/144 phospho-specific antibodyor mass spectrometry (Liu et al., Cell 108 P 837-47 and Gerber et al.,PNAS 100, P 6940-5).

The diagnostic methods described herein can identify subjects having, orat risk of developing, a disease or disorder associated with aberrantHom expression or activity.

The prognostic assays described herein can be used to determine whethera subject is suitable to be administered with an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a cancer. For example,such assays can be used to determine whether a subject can beadministered with a cytotoxic drug to treat a cell proliferationdisorder.

Also featured is a method of monitoring a treatment for a cancer in asubject. For this purpose, gene expression levels of Hom can bedetermined for test samples from a subject before, during, or afterundergoing a treatment. An increase of the expression level of Hom afterthe treatment indicates that the subject can be further treated by thesame treatment. Ser140/144 phosphorylation of an exogenous Xom peptidecan also be used as a monitor of cancers, degenerative diseases andinflammatory/autoimmune diseases activities. For example, one can use aXom protein or its fragment containing the Ser140/144 phosphrylationsite as a substrate, take cells from a subject, and make a cellularextract. Then, the Xom polypeptide is incubated with the extract andmonitoring Ser140/144 phosphorylation with Ser140/144 phospho specificantibody. Alternatively, one can express a Xom polypeptide or itsfragment having Ser140/144 in a cell obtained from a subject anddetermine the Ser140/144 phosphorylation level. The level reflects astage of the disorder based on the teaching provided herein and thatknown in the art.

Information obtained from practice of the above diagnostic assays isuseful in prognostication, identifying progression of, and clinicalmanagement of diseases and other deleterious conditions affecting anindividual's health status. In preferred embodiments, the foregoingdiagnostic assays provide information useful in prognostication,identifying progression of and management of malignancies (cancers) thatare characterized by lack or abnormal low level Hom expression. Theinformation more specifically assists the clinician in designingchemotherapeutic or other treatment regimes to eradicate suchmalignancies from the body of an afflicted mammal, typically a human.

Drug Screening

The invention features a method for identifying a compound that enhancesthe activity of Hom/Xom by inducing its expression or promote itsstability via, e.g., inhibiting phosphorylation of Ser140/144. Thecompound thus-identified can be used to treat cancer, degenerativedisorders, or immune disorders.

A compound that reduces Xom/Hom protein phosphorylation level in astatistically significant manner (i.e., Xom/Hom kinase inhibitor) can beidentified according to the methods described below.

Candidate compounds to be screened (e.g., proteins, peptides,peptidomimetics, peptoids, antibodies, small molecules, or other drugs)can be obtained using any of the numerous approaches in combinatoriallibrary methods known in the art. Such libraries include: peptidelibraries, peptoid libraries (libraries of molecules having thefunctionalities of peptides, but with a novel, non-peptide backbone thatis resistant to enzymatic degradation); spatially addressable parallelsolid phase or solution phase libraries; synthetic libraries obtained bydeconvolution or affinity chromatography selection; and the “one-beadone-compound” libraries. See, e.g., Zuckermann et al. 1994, J. Med.Chem. 37:2678-2685; and Lam, 1997, Anticancer Drug Des. 12:145. Examplesof methods for the synthesis of molecular libraries can be found in,e.g., DeWitt et al., 1993, PNAS USA 90:6909; Erb et al., 1994, PNA USA91:11422; Zuckermann et al., 1994, J. Med. Chem. 37:2678; Cho et al.,1993, Science 261:1303; Carrell et al., 1994, Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2061;and Gallop et al., 1994 J. Med. Chem. 37:1233. Libraries of compoundsmay be presented in solution (e.g., Houghten, 1992, Biotechniques13:412-421), or on beads (Lam, 1991, Nature 354:82-84), chips (Fodor,1993, Nature 364:555-556), bacteria (U.S. Pat. No. 5,223,409), spores(U.S. Pat. No. 5,223,409), plasmids (Cull et al., 1992, PNAS USA89:1865-1869), or phages (Scott and Smith 1990, Science 249:386-390;Devlin, 1990, Science 249:404-406; Cwirla et al., 1990, PNAS USA87:6378-6382; Felici 1991, J. Mol. Biol. 222:301-310; and U.S. Pat. No.5,223,409).

To identify a Xom/Hom activator or a Xom/Hom kinase inhibitor, one cancontact a candidate compound with a system containing a Xom/Hom gene orpolypeptide. The system can be a cell-free system or a cell-containingsystem, e.g., an in vitro cell line model or an in vivo animal model. Ina cell-containing system, cells can naturally express the Xom/Hom gene,or can be modified to express a recombinant nucleic acid. Therecombinant nucleic acid can contain the Xom/Hom gene coding regionfused to a heterologous promoter or a Xom/Hom gene promoter sequencefused to a reporter gene. One then measures the expression level or thephosphorylation level of the Xom/Hom polypeptide (e.g., that at Ser 140or Ser 144). A Xom/Hom polypeptide described above can be a full-lengthXom/Hom polypeptide or its fragment that contains the phosphorylationsites.

The expression level can be determined at either the mRNA level or atthe protein level. Methods of measuring mRNA levels in a cell, a tissuesample, or a body fluid are well known in the art. To measure mRNAlevels, cells can be lysed and the levels of mRNA in the lysates or inRNA purified or semi-purified from the lysates can be determined by,e.g., hybridization assays (using detectably labeled gene-specific DNAor RNA probes) and quantitative or semi-quantitative RT-PCR (usingappropriate gene-specific primers). Alternatively, quantitative orsemi-quantitative in situ hybridization assays can be carried out usingtissue sections or unlysed cell suspensions, and detectably (e.g.,fluorescent or enzyme) labeled DNA or RNA probes. AdditionalmRNA-quantifying methods include RNA protection assay (RPA) and SAGE.Methods of measuring protein levels in a cell or a tissue sample arealso known in the art.

Methods of measuring the phosphorylationlevel of a polypeptide are alsoknown in the art. Examples of the methods include using specificphospho-antibodies or mass spectrometry (Liu et al., Cell 108 P 8347-47and Gerber et al., PNAS 100, P 6940-5).

To determine the ability of a candidate compound to increase Xom/Homexpression level or inhibit its phosphorylation level, one compares thelevel obtained in the manner described above with a control level oractivity obtained in the absence of the candidate compound. If thephosphorylation level is lower than the control, or if the expressionlevel is higher than the control, the compound is identified as beingeffective for treating the disorders mentioned above. One can furtherverify the efficacy of a compound thus-identified using a Xenopus occytemodel or an animal mode. One can administer the compound to Xenopusoocyte model or an animal models and exam them according to the methoddescribe below in the Example section or other standard techniques. Anystatistically significant increase in cell death indicates the compoundis a candidate for treating the disorders mentioned above.

Inhibitors of Xom/Hom can also be identified using the above-describedmethods except that a candidate compound is identified if it repress theexpression of Xom/Hom or increase the phosphorylation level.

Treatment Methods

The invention also features methods for treating in a subject a cellularproliferative disorders (e.g., cancer), a cellular degenerativedisorder, an inflammation-related disorder (eczema and inflammatorybowel diseases), or a hematological condition (e.g., myelodysplasticsyndromes).

A cellular proliferative disorder refers to a disorder characterized byuncontrolled, autonomous cell growth, including malignant andnon-malignant growth. Examples of this disorders include colon cancer,breast cancer, prostate cancer, hepatocellular careinoma, melanoma, lungcancer, glioblastoma, brain tumor, hematopoeitic malignancies,retinoblastoma, renal cell carcinoma, head and neck cancer, cervicalcancer, pancreatic cancer, esophageal cancer, and squama cell carcinoma.

An inflammation-related disorder is characterized by a local orsystemic, acute or chronic inflammation. Examples include inflammatorydermatoses (e.g., dermatitis, eczema, atopic dermatitis, allergiccontact dermatitis, urticaria, necrotizing vasculitis, cutaneousvasculitis, hypersensitivity vasculitis, eosinophilic myositis,polymyositis, dermatomyositis, and eosinophilic fasciitis), inflammatorybowel diseases (e.g., Crohn's disease and ulcerative colitis), acuterespiratory distress syndrome, fulminant hepatitis, hypersensitivitylung diseases (e.g., hypersensitivity pneumonitis, eosinophilicpneumonia, delayed-type hypersensitivity, interstitial lung disease orILD, idiopathic pulmonary fibrosis, and ILD associated with rheumatoidarthritis), asthma, and allergic rhinitis. Examples also includeautoimmune diseases (e.g., rheumatoid arthritis, psoriatic arthritis,systemic lupus erythematosus, myasthenia gravis, juvenile onsetdiabetes, glomerulonephritis, autoimmune throiditis, ankylosingspondylitis, systemic sclerosis, and multiple sclerosis), acute andchronic inflammatory diseases (e.g., systemic anaphylaxia orhypersensivity responses, drug allergies, insect sting allergies,allograft rejection, and graft-versus-host disease), Sjogren's syndrome,human immunodeficiency virus infection, cancer (e.g., brain, breast,prostate, colon, kidney, ovary, thyroid, lung, and hematopoieticcancer), and tumor metastasis.

A “subject” refers to a human and a non-human animal. Examples of anon-human animal include all vertebrates, e.g., mammals, such asnon-human primates (particularly higher primates), dog, rodent (e.g.,mouse or rat), guinea pig, cat, and non-mammals, such as birds,amphibians, reptiles, etc. In a preferred embodiment, the subject is ahuman. In another embodiment, the subject is an experimental animal oranimal suitable as a disease model.

A subject to be treated for a cellular proliferative disorder can beidentified by standard diagnosing techniques for the disorder.Optionally, the subject can then be examined for the gene expression oractivity level of the Xom/Hom gene or polypeptide by methods describedabove. If the gene expression or activity level is lower in a samplefrom the subject than that in a sample from a normal person, the subjectis a candidate for treatment with an effective amount of a Xom/Hompeptide or activator. A subject to be treated for aninflammation-related disorder can be identified by standard diagnosingtechniques.

“Treating” refers to administration of a compound to a subject, who hasa cellular proliferative disorder (e.g., cancer), aninflammation-related, or a hematological condition, with the purpose tocure, alleviate, relieve, remedy, prevent, or ameliorate the disorder,they symptom or the disorder. An “effective amount” refers to an amountof the compound that is capable of producing a medically desirableresult, e.g., as described above, in a treated subject. The treatmentmethod can be preformed in vivo or ex vivo, alone or in conjunction withother drugs or therapy.

In an in vivo approach, a compound is administered to a subject.Generally, the compound is suspended in a pharmaceutically-acceptablecarrier (e.g., physiological saline) and administered orally or byintravenous infusion, or injected or implanted subcutaneously,intramuscularly, intrathecally, intraperitoneally, intrarectally,intravaginally, intranasally, intragastrically, intratracheally, orintrapulmonarily.

The dosage required depends on the choice of the route ofadministration; the nature of the formulation; the nature of thepatient's illness; the subject's size, weight, surface area, age, andsex; other drugs beings administered; and the judgment of the attendingphysician. Suitable dosages are in the range of 0.01-100 mg/kg.Variation in the needed dosage are to be expected in view of the varietyof compounds availabel and the different efficiencies of various routesof administration. For example, oral administration would be expected torequire higher dosages than administration by i.v. injection. Variationsin theses dosage levels can be adjusted using standard empiricalroutines for optimization as is well understood in the art.Encapsulation of the compound in a suitable delivery vehicle (e.g.,polymeric microparticles or implantable devices) may increase theefficiency of delivery, particularly for oral delivery.

Examples of compounds that can be used to treat a cellular proliferativedisorder or an inflammation-related disorder include polypeptides thathave SEQ ID NO: 1 or 3 and lack the LEF1/TCF transactivation domain. Theexamples also include functional equivalents of SEQ ID NO: 1 or 3. Asdescribed above, a functional equivalent of SEQ ID NO: 1 or 3 refers toa polypeptide having one or more point mutations, insertions, deletions,truncations, or combination thereof. This polypeptide is at least 60%(any number between 60% and 100%, inclusive) identical to SEQ ID NO: 1or 3 and retains substantially activity of the homeodomain activity ofXom or Hom, i.e., the ability to bind to LEF1/TCF and inhibitLEF1/TCF-dependent transcription in a dominant negative manner bycompeting for endogenous LEF1/TCF activator, such as β-catenin. LEF1/TCFfactors are essential high mobility group (HMG) containingtranscriptional factors. The LEF1/TCFs contains little transcriptionalactivity by themselves, rather LEF1/TCFs are activated by associatedfactors, such as the β-catenin of the Wnt pathway and Xom of the BMP4pathway. Transactivation of LEF1/TCFs by the β-catenin or Xom has beenfound to be essential for stem cells function and cell fatedetermination as well as malignant transformation.

The aforementioned polypeptides can be synthesized using methods knownin the art or be prepared using recombinant technology. For example, onecan clone a nucleic acid encoding the polypeptide in an expressionvector, in which the nucleic acid is operably linked to a regulatorysequence suitable for expressing the polypeptide in a host cell. One canthen introduce the vector into a suitable host cell to express thepolypeptide. The expressed recombinant polypeptide can be purified fromthe host cell by methods such as ammonium sulfate precipitation andfractionation column chromatography. See Goeddel, (1990) Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.A polypeptide thus prepared can be tested for its activity according tothe method described in the examples below.

Examples of compounds that can be used to treat cellular proliferativedisorders include those that increase the protein level of Hom, such as5-FU, DOX, radiation, retinoic acid, GM-CSF-IL4), resveratrol, ellagicacid, aspirin, salicylic acid, emodin and flavonoid and theirderivatives that induce Hom expression. Also can be used are those thatinhibit Xom/Hom's phosphorylation (i.e., Xom/Hom kinase inhibitors),such as resveratrol, ellagic acid, aspirin, salicylic acid, emodin andflavonoid and their derivatives.

Compounds that inhibit the expression or activity of Hom/Xom can also beuse to treat other disorders, such as Myelodysplastic syndromes (MDS).MDS, also known as “preleukemia,” are a diverse collection ofhematological conditions united by ineffective production of blood cellsand varying risks of transformation to acute myelogenous leukemia.Although not a true malignant neoplasm, MDS is nevertheless classifiedwithin the hematological neoplasms. It was found that Hom wasover-expressed in an MDS patient, suggesting the blockage of thedifferentiation progress by Hom. Thus, an inhibitor of Hom/Xom can beused to treat MDS. Examples of the inhibitor include an antibody, andantisense nucleic acid, and an RNAi agent that specifically targetHom/Xom. Other examples include compounds that inhibit the expression ofHom.

An “antibody” includes intact molecules as well as fragments thereof,such as Fab, F(ab′)2, Fv, scFv (single chain antibody), and dAb (domainantibody; Ward, et al. (1989) Nature, 341, 544). A derivative of anantibody refers to a protein or a protein complex having a polypeptidevariant of this invention. An antibody or derivative of this inventioncan be made by co-expressing corresponding light and heavy chainCDRs-containing polypeptides in a suitable host cell by methods known inthe art. See, e.g., Harlow and Lane, (1988) Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, New York.

To make an antibody described herein, the Xom or Hom polypeptide or itsantigenic fragment can be coupled to a carrier protein, such as KLH,mixed with an adjuvant, and injected into a host animal. Antibodiesproduced in that animal can then be purified by peptide affinitychromatography. Commonly employed host animals include rabbits, mice,guinea pigs, and rats. Various adjuvants that can be used to increasethe immunological response depend on the host species and includeFreund's adjuvant (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, and dinitrophenol. Useful human adjuvants include BCG(bacille Calmette-Guerin) and Corynebacterium parvum.

Polyclonal antibodies, heterogeneous populations of antibody molecules,are present in the sera of the immunized subjects. Monoclonalantibodies, homogeneous populations of antibodies to a particularantigen, can be prepared using standard hybridoma technology. See, e.g.,Kohler et al. (1975) Nature 256, 495; Kohler et al. (1976) Eur. J.Immunol. 6, 511; Kohler et al. (1976) Eur. J. Immunol. 6, 292; andHammerling et al. (1981) Monoclonal Antibodies and T Cell Hybriodomas,Elsevier, N.Y. In particular, monoclonal antibodies can be obtained byany technique that provides for the production of antibody molecules bycontinuous cell lines in culture such as described in U.S. Pat. No.4,376,110; the human B-cell hybridoma technique (Kosbor et al. (1983)Immunol Today 4, 72; Cole et al. (1983) Proc. Natl. Acad. Sci. USA 80,2026) and the EBV hybridoma technique (Cole et al. (1983) MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Suchantibodies can be of any immunoglobulin class including IgG, IgM, IgE,IgA, IgD, and any subclass thereof. The hybridoma producing themonoclonal antibodies of the invention may be cultivated in vitro and invivo. The ability to produce high titers of monoclonal antibodies invivo makes it a particularly useful method of production.

A polynucleotide containing a nucleic acid sequence encoding aninhibitor of Xom or Hom can be used to treat an inflammation-relateddisorder. The nucleic acid sequence can encode the above-describedpolypeptide, an anti-Xom or Hom antibody, and anti-sense RNA, or a smallinterference RNA (e.g., and RNAi agent) that targets the Xom or Hom andinhibits its expression activity.

The term “RNAi” or “RNA interference” refers to a sequence-specific orselective process by which a target molecule (e.g., a target gene,protein or RNA) is down-regulated. Within the scope of this invention isutilization of RNAi featuring degradation of RNA molecules (e.g., withina cell). Degradation is catalyzed by an enzymatic, RNA-induced silencingcomplex (RISC). RNAi occurs in cells naturally to remove foreign RNAs(e.g., viral RNAs). Natural RNAi proceeds via fragments cleaved fromfree double-stranded RNA, which directs the degradative mechanism.Alternatively, RNAi can be initiated by the hand of man, for example, tosilence the expression of target genes.

The term “RNAi agent” refers to an RNA (or analog thereof), havingsufficient sequence complementarity to a target RNA (i.e., the RNA beingdegraded) to direct RNAi. A RNA agent having a “sequence sufficientlycomplementary to a target RNA sequence to direct RNAi” means that theRNA agent has a sequence sufficient to trigger the destruction of thetarget RNA by the RNAi machinery (e.g., the RISC complex) or process. ARNA agent having a “sequence sufficiently complementary to a target RNAsequence to direct RNAi” also means that the RNA agent has a sequencesufficient to trigger the translational inhibition of the target RNA bythe RNAi machinery or process. A RNA agent can also have a sequencesufficiently complementary to a target RNA encoded by the target DNAsequence such that the target DNA sequence is chromatically silenced. Inother words, the RNA agent has a sequence sufficient to inducetranscriptional gene silencing, e.g., to down-modulate gene expressionat or near the target DNA sequence, e.g., by inducing chromatinstructural changes at or near the target DNA sequence. The term “RNA” or“RNA molecule” or “ribonucleic acid molecule” refers to a polymer ofribonucleotides. The term “DNA” or “DNA molecule” or deoxyribonucleicacid molecule” refers to a polymer of deoxyribonucleotides. DNA and RNAcan be synthesized naturally (e.g., by DNA replication or transcriptionof DNA, respectively), RNA can be post-transcriptionally modified. DNAand RNA can also be chemically synthesized. DNA and RNA can besingle-stranded (i.e., ssRNA and ssDNA, respectively) or multi-stranded(e.g., double-stranded, i.e., dsRNA and dsDNA, respectively).

The polynucleotide can be delivered by the use of polymeric,biodegradable microparticle or microcapsule delivery devices known inthe art. Another way to achieve uptake of the nucleic acid is usingliposomes, prepared by standard methods. The polynucleotide can beincorporated alone into these delivery vehicles or co-incorporated withtissue-specific antibodies. Alternatively, one can prepare a molecularconjugate composed of a plasmid or other vector attached topoly-L-lysine by electrostatic or covalent forces. Poly-L-lysine bindsto a ligand that can bind to a receptor on target cells (Cristiano, etal., 1995, J. Mol. Med. 73:479). Alternatively, tissue specifictargeting can be achieved by the use of tissue-specific transcriptionalregulatory elements that are known in the art. Delivery of “naked DNA”(i.e., without a delivery vehicle) to an intramuscular, intradermal, orsubcutaneous site is another means to achieve in vivo expression.

In the above-mentioned polynucleotides, e.g., expression vectors, thenucleic acid sequence encoding an inhibitor of Xom or Hom is operativelylinked to a promoter or enhancer-promoter combination. Suitableexpression vectors include plasmids and viral vectors such as herpesviruses, retroviruses, vaccinia viruses, attenuated vaccinia viruses,canary pox viruses, adenoviruses and adeno-associated viruses.

As is well known in the art, the dosage for a patient depends uponvarious factors as described above. Dosages will vary, but a preferreddosage for administration of polynucleotide is about 10⁶ to 10¹² copiesof the polynucleotide molecule. This dose can be repeatedly administeredas needed. Routes of administration can be any of those listed above.

Also within the scope of the invention is a packaged product including acontainer, an effective amount of one of the above-described compoundand a legend associated with the container and indicating administrationof the compound for treating a subject suffering from or being at riskfor developing the disorder mentioned above. The compound can be admixedwith a pharmaceutically acceptable carrier, including a solvent, adispersion medium, a coating, an antibacterial and antifungal agent, andan isotonic and absorption-delaying agent.

The compound can be formulated into dosage forms for differentadministration routes utilizing conventional methods. For example, itcan be formulated in a capsule, a gel seal, or a tablet for oraladministration. Capsules can contain any standard pharmaceuticallyacceptable materials such as gelatin or cellulose. Tablets can beformulated in accordance with conventional procedures by compressingmixtures of the compound with a solid carrier and a lubricant. Examplesof solid carriers include starch and sugar bentonite. The compound canalso be administered in a form of a hard shell tablet or a capsulecontaining a binder, e.g., lactose or mannitol, a conventional filler,and a tableting agent. The compound can be administered via theparenteral route. Examples of parenteral dosage forms include aqueoussolutions, isotonic saline or 5% glucose of the active agent, or otherwell-known pharmaceutically acceptable excipient. Cyclodextrins, orother solubilizing agents well known to those familiar with the art, canbe utilized as pharmaceutical excipients for delivery of the therapeuticagent.

The efficacy of the compound can be evaluated both in vitro and in vivo.For example, the compound can be tested for its ability to arrest cellgrowth or induce apoptosis in vitro. For in vivo studies, the compoundcan be injected into an animal (e.g., an animal model) and its effectson cells growth or apoptosis are then accessed. Based on the results, anappropriate dosage range and administration route can be determined.

Stem Cells

A stem cell is a cell that is capable of differentiating into a numberof final, differentiated cells types. Stem cells may be totipotent,pluripotent, mulitpotent, or unipotent. Totipotent stem cells areproduced from the fusion of an egg and sperm cell. Cells produced by thefirst few divisions of the fertilized egg are also totipotent. Thesecells can differentiate into embryonic and extraembryonic cell types.Totipotent stem cells typically have the capacity to develop into anycell type. totipotent stem cells are usually embryonic in origin.Pluripotent stem cells are the descendants of totipotent cells and candifferentiate into cells derived from the three germ layers. These cellsare typically cells in a stem cell line capable of differentiating intoseveral different, final differentiated cells types. Multipotent stemcells can produce only cells of a closely related family of cells (e.g.,hematopoietic stem cells differentiate into red blood cells, white bloodcells, platelets, etc.). Unipotent cells can produce only once celltype, but have the property of self-renewal which distinguishes themfrom non-stem cells. Pluripotent, multipotent, and unipotent stem cellscan originate from various tissue or organ systems, including, but notlimited to, blood, nerve, muscle, skin, gut, bone, kidney, liver,pancreas, thymus, and the like.

As mentioned above, the Wnt and BMP signaling pathways are involved inthe proliferation or differentiation of various stem cells. See e.g.,Reya et al., Nature . 2005 Apr. 14; 434(7035):843-50. As a keycomponent, Xom/Hom plays an important role in the preservation,expansion, and differentiation of the stem cells. Thus, Xom/Hom can beused in regulating development and differentiation of the stem cells,and in treating related proliferative or degenerative diseases. Forexample, Xom/Hom may functioning slowing down or preventingdifferentiation of stem cells and, thereby, maintains a stem cell pool.It follows that an antagonist (i.e., inhibitor) of Xom/Hom can be usedto promoting differentiation.

Dysregulation of this differentiation process results in variousdisorders, such as MDS. MDS are thought to arise from mutations in themulti-potent bone marrow stem cell. Differentiation of blood precursorcells is impaired, and there is a significant increase in levels of celldeath apoptosis in bone marrow cells. Clonal expansion of the abnormalcells results in the production of cells which have lost the ability todifferentiate. The progression of MDS to leukemia is a good example ofthe multi-step theory of carcinogenesis in which a series of mutationsoccur in an initially normal cell and transform it into a cancer cell.As described above, over expression of Hom/Xom in a MDS patient may leadto the blockage of the differentiation progress and apoptosis seen inMDS. Thus, an inhibitor of Hom/Xom can be used to treat MDS. Examples ofthe inhibitor include antibodies, antisense nucleic acids, RNAi agents,and small molecular compounds that specifically target Hom/Xom.

The treatment method of this invention can be used to treat theMyelodysplastic syndromes by promoting cell differentiation.

The method of this invention can also be used to treat or slow down theprogress of other proliferative or degenerative disorders. Examples ofthe disorders include macular degeneration, neuron degeneration,Huntington's Disease, Parkinson's Disease, Alzheimer's Disease and,Schizophrenia.

The terms “proliferation” and “expansion” as used interchangeably hereinwith reference to cells, refer to an increase in the number of cells ofthe same type by division. The term “differentiation” refers to adevelopmental process whereby cells become specialized for a particularfunction, for example, where cells acquire one or more morphologicalcharacteristics and/or functions different from that of the initial celltype. The term “differentiation” includes both lineage commitment andterminal differentiation processes. Differentiation may be assessed, forexample, by monitoring the presence or absence of lineage markers, usingimmunohistochemistry or other procedures known to a worker skilled inthe art. Differentiated progeny cells derived from progenitor cells maybe, but are not necessarily, related to the same germ layer or tissue asthe source tissue of the stem cells. For example, neural progenitorcells and muscle progenitor cells can differentiate into hematopoieticcell lineages.

The terms “lineage commitment” and “specification,” as usedinterchangeably herein, refer to the process a stem cell undergoes inwhich the stem cell gives rise to a progenitor cell committed to forminga particular limited range of differentiated cell types. Committedprogenitor cells are often capable of self-renewal or cell division.

The term “terminal differentiation,” as used herein, refers to the finaldifferentiation of a cell into a mature, fully differentiated cell. Forexample, neural progenitor cells and muscle progenitor cells candifferentiate into hematopoietic cell lineages, terminal differentiationof which leads to mature blood cells of a specific cell type. Usually,terminal differentiation is associated with-withdrawal from the cellcycle and cessation of proliferation. The term “progenitor cell,” asused herein, refers to a cell that is committed to a particular celllineage and which gives rise to cells of this lineage by a series ofcells division. An example of a progenitor cell would be a myoblast,which is capable of differentiation to only one type of cell, but isitself not fully mature of fully differentiated.

Compositions

Within the scope of this invention is a composition that contains asuitable carrier and one or more of the compounds described above. Thecomposition can be a pharmaceutical composition that contains apharmaceutically acceptable carrier, a dietary composition that containsa dietarily acceptable suitable carrier, or a cosmetic composition thatcontains a cosmetically acceptable carrier.

Examples of a dietary composition of the present invention include anactive compound described above. Examples of this compound includeHom/Xom polypeptide or a functional fragment thereof, retinoic acid,resveratrol, ellagic acid, aspirin, salicylic acid, emodin, andflavonoid, and derivatives of these compounds. The composition alsoincludes, but is not limited to, foods, food additives, nutritionalsupplements, and pharmaceutical preparations. It may be in the form oftablets, suspensions, implants, solutions, emulsions, capsules, powders,syrups, liquid composition, ointments, lotions, creams, pastes, gels, orthe like.

As a dietary supplement, additional nutrients, such as minerals or aminoacids, may be included. A dietary composition can also be a drink orfood product. As used herein, the terms“drink” and “food” broadly referto any kinds of liquid and solid/semi-solid materials, respectively,that are used for nourishing an animal, and for sustaining normal oraccelerated growth of an animal including a human. Examples of the drinkproduct include, but are not limited to, tea-based beverages, juice,coffee, and milk. Examples of the food product include jelly, cookies,cereals, chocolates, snack bars, herbal extracts, dairy products (e.g.,ice cream, and yogurt), soy bean product (e.g., tofu), and riseproducts.

A composition of the present invention may include a carrier. Dependingon the kind of the composition, a carrier may be a suitable dietarycarrier or a pharmaceutically acceptable carrier. Examples of apharmaceutically acceptable carrier include, but are not limited to,biocompatible vehicles, adjuvants, additives, and diluents to achieve acomposition usable as a dosage form.

A “pharmaceutically acceptable carrier,” after administered to or upon asubject, does not cause undesirable physiological effects. The carrierin the pharmaceutical composition must be “acceptable” also in the sensethat it is compatible with the active ingredient and, preferably,capable of stabilizing it. One or more solubilizing agents can beutilized as pharmaceutical carriers for delivery of an active compound.Examples of other carriers include colloidal silicon oxide, magnesiumstearate, cellulose, sodium lauryl sulfate, and D&C Yellow #10.

The above-described composition, in any of the forms described above,can be used for treating cellular proliferative disorders andinflammation-related disorders.

An “effective amount” refers to the amount of an active compound that isrequired to confer a therapeutic effect on a treated subject. Effectivedoses will vary, as recognized by those skilled in the art, depending onthe types of diseases treated, route of administration, excipient usage,and the possibility of co-usage with other therapeutic treatment.

A pharmaceutical composition of this invention can be administeredparenterally, orally, nasally, rectally, topically, or buccally, Theterm “parenteral” as used herein refers to subcutaneous, intracutaneous,intravenous, intramuscular, intraarticular, intraarterial,intrasynovial, intrasternal, intrathecal, intralesional, or intracranialinjection, as well as any suitable infusion technique.

A sterile injectable composition can be a solution or suspension in anon-toxic parenterally acceptable diluent or solvent, such as a solutionin 1,3-butanediol. Among the acceptable vehicles and solvents that canbe employed are mannitol, water, Ringer's solution, and isotonic sodiumchloride solution. In addition, fixed oils are conventionally employedas a solvent or suspending medium (e.g., synthetic mono- ordiglycerides). Fatty acid, such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as are naturalpharmaceutically acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions can also contain a long chain alcohol diluent or dispersant,carboxymethyl cellulose, or similar dispersing agents. Other commonlyused surfactants such as Tweens or Spans or other similar emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms can also be used for the purpose of formulation.

A composition for oral administration can be any orally acceptabledosage form including capsules, tablets, emulsions and aqueoussuspensions, dispersions, and solution. In the case of tablets, commonlyused carriers include lactose and corn starch. Lubricating agents, suchas magnesium stearate are also typically added. For oral administrationin a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions or emulsions are administered orally,the active ingredient can be suspended or dissolved in an oily phasecombined with emulsifying or suspending agents. If desired, certainsweetening, flavoring, or coloring agents can be added.

A nasal aerosol or inhalation composition can be prepared according totechniques well known in the art of pharmaceutical formulation. Forexample, such a composition can be prepared as s solution in saline,employing benzyl alcohol or other suitable preservatives, absorptionpromoters to enhance bioavailability, fluorocarbons, and/or othersolubilizing or dispersing agents known in the art.

A composition having an active compound can also be administered in theform of suppositories for rectal administration.

A topical composition contains a sfe and effective amount of adermatologically acceptable carrier suitable for application to theskin. Generally, a topical composition can be solid, semi-solid, creamliquid. It may be a cosmetic or dermatologic product in the form of anointment, lotion, foam, cream gel, or solution. Details aboutdermatologically acceptable carriers are provided below.

A composition of the present invention may be used alone or incombination with other biologically active ingredients. Alone or incombination with other active ingredients, it may be administered to asubject in a single dose or multiple doses over a period of time.Various administration patterns will be apparent to those skilled in theart. The dosage ranges for the administration of the composition arethose large enough to produce the desired effect. The dosage should notbe so large as to cause any adverse side effects, such as unwantedcross-reactions and the like. Generally, the dosage will vary with theage, weight, sex, condition, and extent of a condition in a subject, andthe intended purpose. The dosage can be determined by oneof skill in theart without undue experimentation. The dosage can be adjusted in theevent of any counter indications, tolerance, or similar conditions.Those of skill in the art can readily evaluate such factors and, basedon this information, determine the particular effective concentration ofa composition of the present invention to be used for an intendedpurpose.

Also within the scope of this invention is a cosmetic composition thatcontains an active compound described above. Examples of this compoundinclude Hom/Xom polypeptide or a functional fragment thereof. Examplesalso include retinoic acid, resveratrol, ellagic acid, aspirin,salicylic acid, emodin, and flavonoid, and derivatives of thesecompounds. This composition contains a safe and effective amount of adermatologically acceptable carrier that is suitable for topicalapplication to the skin. It enables an active compound and optionalcomponent to be delivered to the skin at an appropriateconcentrations(s). The carrier can thus act as a diluent, dispersant,solvent, or the like to ensure that the active materials are applied toand distributed evenly over the selected target at an appropriateconcentration. The carrier can be solid, semi-solid, or liquid.Preferably, it is in the form of a lotion, a cream, or a gel, inparticular one that has a sufficient thickness or yield point to preventthe active materials from sedimenting. The carrier can be inert orpossess dermatological benefits of its own. It should also be physicallyand chemically compatible with the active components described herein,and should not unduly impair stability, efficacy, or other use benefitsassociated with the composition.

The type of carrier utilized in the cosmetic composition depends on thetype of product form of the composition. a cosmetic composition may bemade into a wide variety of product forms such as those known in theart. These include, but are not limited to, lotions, creams, gels,sprays, ointments, pastes, and mousses. These product forms may compriseseveral types of carriers including, but not limited to, solutions,aerosols, emulsions, gels, solids, and liposomes.

Preferred carriers can contain a dermatologically acceptable,hydrophilic diluent. Suitable hydrophilic diluents include water,organic hydrophilic diluents, such as C₁-C₄ monohydric alcohols and lowmolecular weight glycols and polyols (includes propylene glycol,polyethylene glycol of, e.g., MW 200-600), polypropylene glycol of,e.g., MW 425-2025, glycerol, butylene glycol, 1,2,4-butanetriol,sorbitol esters, 1,2,6-hexanetriol, ethanol, iso-propanol, sorbitolesters, ethoxylated ethers, propoxylated ethers, and combinationsthereof. The composition preferably comprises at least about 60% of thehydrophilic diluent.

Preferred carriers also contain an emulsion having a hydrophilic phase,especially an aqueous phase, and a hydrophobic phase, e.g., a lipid oil,or oily material. As well known to one skilled in the art, thehydrophilic phase will be dispersed in the hydrophobic phase, or viceversa, to form respectively hydrophilic or hydrophobic dispersed andcontinuous phases, depending on the composition ingredients. The term“dispersed phase,” a term well-known to one skilled in the art, refersto a phase that exists as small particles or droplets suspended in andsurrounded by a continuous phase. The dispersed phase is also known asthe internal or discontinuous phase. The emulsion may be or contain(e.g., in a triple or other multi-phase emulsion) an oil-in-wateremulsion or a water-in-oil emulsion such as a water-in-siliconeemulsion. Oil-in-water emulsions typically comprise from 1% to 50%(preferable 1% to 30%) of the dispersed hydrophobic phase and from 1% to99% (preferably from 40% to 90%) of the continuous hydrophilic phase;water-in-oil emulsions typically comprise 1% to 50% (preferably from 40%to 90%) of the dispersed hydrophilic phase and from 1% to 50%(preferably 1% to 30%) of the continuous hydrophobic phase. The emulsionmay also comprise a gel network, such as that described in G. M.Eccleston, Application of Emulsion Stability Theories to Mobile andSemisolid O/W Emulsions, Cosmetics & Toiletries, Vol. 101, November1996, pp. 73-92, incorporated herein by reference. Preferredcompositions herein are oil-in-water emulsions.

Preferred examples of a cosmetic composition of this invention have anapparent viscosity of from about 5,000 to about 200,000 mPa·s(centipoise). For example, preferred lotions have an apparent viscosityof from about 10,000 to about 40,000 mPa·s; and preferred creams have anapparent viscosity of from about 30,000 to about 160,000 mPa·s. Apparentviscosity can be determined using a Brookfield DVII RV viscometer,spindle TD, at 5 rpm, or the equivalent thereof. The viscosity isdetermined on a composition after the composition has been allowed tostabilize following its preparation, generally at least 24 hours underconditions of 25° C.±1° C. and ambient pressure after preparation of thecomposition. Apparent viscosity is measured with the composition at atemperature of 25° C.±1° C., after 30 seconds spindle rotation.

The cosmetic composition of the present invention is usually formulatedto have a pH of 9.5 or below and in general have a pH in the range from4.5 to 9, more preferably from 5 to 8.5. Some examples, particularlythose containing an additional active agent such as salicylic acid,require a lower pH in order for the additional active to be fullyefficacious. These compositions are usually formulated to have a pH offrom 2.5 to 5, more preferably from 2.7 to 4.

The cosmetic composition may contain a wide variety of optionalcomponents, provided that such optional components are physically andchemically compatible with the essential components described herein,and do not unduly impair stability, efficacy, or other use benefitsassociated with the compositions. Optional components may be dispersed,dissolved, or the like in the carrier of the present compositions.

Exemplary optional components include emollients, oil absorbents,antimicrobial agents, binders, buffering agents, denaturants, cosmeticastringents, external analgesics, film formers, humectants, opacifyingagents, perfumes, pigments, skin soothing and healing agents,preservatives, propellants, skin penetration enhancers, solvents,suspending agents, emulsifiers, cleansing agents, thickening agents,solubilising agents, waxes, sunscreens, sunless tanning agents,antioxidants and/or radical scavengers, chelating agents, anti-acneagents, anti-inflammatory agents, desquamation agents/exfoliants,organic hydroxy acids, vitamins, and natural extracts. Examples of suchmaterials are described in Harry's Cosmeticology, 7th Ed., Harry &Wilkinson (Hill Publishers, London 1982); in Pharmaceutical DosageForms—Disperse Systems; Lieberman, Rieger & Banker, Vols. 1 (1988) & 2(1989); Marcel Decker, Inc., in The Chemistry and Manufacture ofCosmetics, 2nd Ed., deNavarre (Van Nostrand 1962-1965); and in TheHandbook of Cosmetic Science and Technology, 1st Ed. Knowlton & Pearce(Elsevier 1993) can also be used in the present invention.

The cosmetic composition of the present invention is generally preparedby conventional methods known in the art of making topical compositions.Such methods typically involve mixing of the ingredients in one or moresteps to a relatively uniform state, with or without heating, cooling,application of vacuum, and the like.

The cosmetic composition is useful for regulating or improving skincondition, including regulating visible or tactile wrinkles ordiscontinuities in skin, e.g., visible or tactile wrinkles ordiscontinuities in skin texture or color, more especially thoseassociated with skin inflammation, ageing, or other internal factors(e.g., biochemical changes from within the skin) or external factors(e.g., ultraviolet radiation, environmental pollution, wind, heat, lowhumidity, harsh surfactants, and abrasives).

Regulating skin conditions can be carried out prophylactically ortherapeutically. Prophylactical regulation includes delaying,minimizing, or preventing visible or tactile, swellings, wrinkles ordiscontinuities in skin. Therapeutic regulation, on the other hand,includes ameliorating, diminishing, minimizing or effacing suchswellings, wrinkles or discontinuities. Regulating skin conditionsinvolves improving skin appearance feel, e.g., providing a smoother,more even appearance or feel and reducing signs of aging.

A cosmetic composition is topically applied to the skin in a safe andeffective amount. The applied amount, the frequency of application, andthe period of use vary widely depending upon the active levels of agiven composition and the level of regulation desired. Typically, thecomposition can be applied once per day. However application rates canvary from about once per week up to about three times per day or more.

The cosmetic composition of this invention provides visible improvementin skin condition essentially immediately following application of thecomposition to the skin. Such immediate improvement involves covering ormasking skin imperfections such as textural discontinuities (includingthose associated with skin inflammation or aging, e.g., enlarged pores),or providing a more even skin tone or color. The composition alsoprovides visible improvement in skin condition following chronic topicalapplication, e.g., one week, one year, or the subject's life time.

Regulating skin conditions is preferably performed by applying acomposition in the form of a skin lotion, cream, cosmetic, or the likewhich is intended to be left on the skin for an extended period for someaesthetic, prophylactic, therapeutic or other benefits, i.e., a“leave-on” composition. After applying the composition to the skin, the“leave-on” composition is preferably left on the skin for a period of atleast 15 minutes and up to 12 hours.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the scope of thefollowing claims.

The specific examples below are to be construed as merely illustrative,and not limitative of the remainder of the disclosure in any waywhatsoever. Without further elaboration, it is believed that one skilledin the art can, based on the description herein, utilize the presentinvention to its fullest extent. All publications cited herein arehereby incorporated by reference in their entirety. Further, anymechanism proposed below does not in any way restrict the scope of theclaimed invention.

Example 1 Experimental Procedures

Preparation of Xenopus embryos, microinjection, and luciferaseassays—Protocols for handling embryos and for luciferase assays wereessentially as described in Zhu et al., 2002, Dev Cell 3, 557-568. For atypical cellular luciferase assay, 2×10⁵ cells were split into 12-wellcell-culture plates and incubated for 24 hours and then transfected with3 μl of liposome transfection reagent (TransITI, Mirus), 1 μg of DNAplasmids of selected genes, 0.3 μg of reporter plasmids (Hela cellsrequire one third while 293T cells require one sixth of the DNA andliposome amount). At 48 hours post-transfection, the cells were washedwith PBS, lysed with 1× cell lysis buffer (Promega cell lysis buffer),scraped, and collected. After incubation on ice for 30 minutes, thecells were cleared by centrifugation at 12,000 g for 15 seconds andtransferred to a new tube; then 20 μl of the cell lysate was mixed with100 μl of Luciferase Assay reagent (Promega), and the luciferaseactivity was measured by scintillation counting.

Plasmids and recombinant proteins-Xenopus LEF1, LEF1ΔHMG, LEF1Δ60,LEF1Δ160, Xom, and its deletion mutants were subcloned by the polymerasechain reaction base technique into the pCS2+ and PGEX4T3 vectors andverified by in vitro translation and sequencing. pGL3-OT and pGL3-OFwere generous gifts from Dr. B Volgestein; p2.4BMP4-Luc was a generousgift from Dr. J. Feng; pGL3 promoter MSX2 (WT) and pGL3 promoterMSX2-SDM-600/-766 were generous gifts from Dr. C Sirard.

Preparation of nuclear and cytoplasmic extracts—Cells (4×10⁶) weretrypsinized and washed twice with PBS and pelleted by centrifugation.Total protein was obtained by lysing the cells in 150 μl of RIPA buffer(150 mM NaCL, 1% NP40, 0.5% DOC, 0.1% SDS, 50 mM Tris pH8.0) containing1× proteinase inhibitors (Roche, proteinase inhibitor cocktail).Cytoplasmic protein were obtained by incubation of 2×10⁶ cells with 150μl of hypotonic buffer (0.05% NP40, 10 mM HEPES pH7.9, 1.5 mM MgCl₂, 10mM KCL, 1 mM DTT) containing 1× proteinase inhibitors on ice for 10minutes and then centrifuged at 4000 rpm for 2 minutes. The supernatantswere collected and used as cytoplasmic proteins. The nuclear pelletswere washed twice with PBS. Nuclear proteins were obtained by incubatingthe pellet on ice for 60 minutes in 150 μl of RIPA buffer. Levels ofβ-catenin in fractioned cellular extracts were determined by westernblotting using specific antibody (Takara Bio).

GST pull-down assay—Five micrograms of GST fusion proteins and 5 μl of³⁵S-labeled IVT proteins were mixed with 20 μl of 50% glutathioneSepharose 4B beads in 500 μl of binding buffer (50 mM Tris pH 8.0, 100mM NaCl, 50 mM KCL, 5 mM MgCl₂, 1 mM DTT, 10% glycerol and 0.2% NP40).The mixtures were incubated at 4° C. for 3 hours and washed with 1×PBSplus 0.2% NP40 four times. Bound material was released with 2× samplebuffer, boiled at 95° C. for 5 min, centrifuged briefly, and revealed bySDS-PAGE and autoradiography.

Co-immunoprecipitation—The affinity-protein G beads were prepared bymixing 20 μl of protein G plus-Agarose beads (Santa Cruz Biotechnology)with 1 μg of corresponding antibody. A total of 2×10⁶ of cells werelysed with 1× cells lysis buffer (Promega) containing 1× proteaseinhibitor reagent (Roche), incubated on ice for 30 minutes, sonicatedbriefly, and centrifuged at 12,000 g for 5 minutes at 4° C. Thesupernatants were further cleaned with 20 μl of untreated protein Gplus-Agarose beads and 2 μg of pre-immune serum for 2 hours at 4° C. Thesupernatants were then mixed with 20 μl of prelabeled protein Gplus-Agarose beads at 4° C. overnight. The beads were washed four timeswith PBS plus 0.2% NP40. Bound proteins were released by 2× samplebuffer, boiled at 95° C. for 5 minutes centrifuged briefly, and revealedby western-blot analysis with specific antibodies.

Histological staining—Embryos were fixed with 3.7% formaldehyde, 0.1 MMOPS (pH 7.4), 2 mM EGTA, and 1 mM MgSO₂ at indicated stages. Theembryos were then embedded in paraffin and sectioned at a 10-μmthickness. The sections were stained with hematoxylin and eosin (H&E),and subjected to histological analysis. The sections were furtherstained with 2 μg/ml of 4′,6-diamidino-2-phenylindole, dihydrochloride(DAPI) (Invitrogen), to reveal the nucleus of the embryonic cells.

Analysis of gene expression by real-time PCR—Total RNA was extracted byTrisol methods. Eight embryos from each treatment group were pooled andHomogenized in 1 ml of Trizol (Invitrogen). Chloroform (200 μl) was thenadded to the sample. After vortex mixing, the samples were centrifugedat 12,000 rpm for 1 minute and the supernatants were collected in newtubes. Isoproponol (500 μl) was added to each sample, mixed, and kept atRT for 10 minutes. Samples were then centrifuged for 30 minutes. Thepellets were washed twice with 70% ethanol, air dried, and suspended in100 μl of ddH₂O. The final RNA concentration was determined bymeasurement at OD₂₆₀. First-strand cDNA was synthesized with theSuperScript first-strand synthesis system (Invitrogen) according to themanufacturer's protocol. Briefly, 2.5 μg of total RNA from each samplewas used. The final volume of the RT product was 20 μl and was dilutedto 200 μl (10× dilution); 8 μl of the diluted RT product was used forreal-time PCR using LightCycler System (Roche) and LightCycler FastStartDNA Master SYBR Green I, according to manufacturer's instructions. Therelative levels of gene expression were calculated by the formula;relative gene expression=2−ΔCd(Δd=cycle of the specific gene−cycle ofthe reference Histone-4 gene). Each sample was done in triplicate. Thesequences of the primers are available upon request.

Results

Xom transactivates LEF1/TCF-mediated transcription—Xom is a majorventral cell fate determination factor of the BMP4 signaling pathway.Using the TOPflash assay, a previous investigation locatedLEF1/TCF-mediated transcriptional activities in the ventral-posteriorside of embryos (Kiecker et al., 2001, Development 128, 4189-4201. Byinjecting TOPflash plasmid into the two ventral blastomeres at the4-cell stage and examining luciferase activities during gastrulationstage, it was confirmed the LEF1/TCF-mediated transcriptional activitieson the ventral side of embryos during early embryogenesis (the pGL3-OT,which contains three copies of the optimal TCF binding motif, was agenerous gift from Dr. B Vogelstein. To explore a potentialinter-relationship between Xom expression and LEF/TCF-mediatedtranscription was examined using the TOPflash-luciferase assay. WhenmRNA encoding Xom was co-injected with the TOPflash reporter constructinto Xenopus embryos at the two-cell stage, expression of Xom enhancedthe TOPflash reporter transcriptions sevenfold as compared with itsexpression in embryos injected with a construct of TOPflash alone. Thetrans-activating effect of Xom on the TOPflash reporter was specific,since expression of Xom did not activate control FOPflash reporter(pGL3-OF, also a gift from Dr. Vogelstein) which contains mutations atthe LEF1/TCF binding sites). To determine whether Xom transaction ofLEF1/TCF-mediated transcription is context-dependent in embryos, theeffect of Xom expression on LEF1/TCF-mediated transcription innon-embryonic cells was also studied. When plasmids encoding Xom andTOPflash were co-transfected into HeLa and HCT116 cells (and later 293Tcells), Xom expression induced the transcription of the TOPflashconstructs in these non-embryonic mammalian cells, indicating that Xomexerts a general induction effect on LEF1/TCF-mediated transcription.Again, the specificity of Xom transactivates LEF1/TCF-mediatedtranscription was indicated by the finding that Xom failed to activatethe FOPflash reporter construct in these tissue culture experiments.

Xom binds to LEF1/TCF factors in vivo and in vitro—Binding of LEF1/TCFsto the promoter of TOPflash is required for the activation of thereporter construct; thus, the finding that expression of Xom activatesTOPflash but not FOPflash raised the possibility that Xom activates theTOPflash reporter through interaction with LEF/TCFs. This hypothesis wastested by determining whether Xom physically interacts with LEF1/TCFs.Co-immunoprecipitation experiments was carried out in HCT116 cellstransiently transfected with myc-Xom. It was found thatimmunoprecipitation of endogenous TCF4 with anti-TCF4 antibodiesco-precipitated the Xom protein. The in vivo association between Xom andTCF4 appears to be strong and could not be dissociated with 300 mM NaCland 0.1% NP40. To determine the domain of Xom that mediates itsinteraction with LEF1/TCFs, serial deletion mutants of Xom were made andtested for their interactions with TCF4 in vivo. The proteins used were:wild type Xom (aa 1-326), XomND55 (aa. 56-326), Xom ND175 (aa. 176-326),XomCD145 (aa. 1-181), XomCD85 (aa. 1-241), and XomID110 (fusion of aa1-130 and aa 241-326). All Xom mutants that carry the Homeobox regionco-immuno-precipitated with anti-TCF4 beads, while those lacking theHomeodomain did not.

These data indicate that the Homeodomain of Xom plays a critical role inmediating complex formation between Xom and LEF1/TCF factors. Followingthe demonstration of complex formation between Xom and LEF1/TCF factors.Following the demonstration of complex formation between Xom and TCF4 invivo, the critical domains of LEF1/TCFs involved in the interaction withXom were identified. At least four members of the LEF1/TCF family havebeen identified in vertebrates (LEF1, TCF1, TCT3, and TCF4), and LEF1shows an expression pattern similar to that of Xom. Zygotictranscription of LEF1 starts after the onset of the MBT and is enrichedat the ventral-caudal side of the animal (Molenaar et al., 1998, MechDev 75, 151-154). Genetic data indicate that LEF1 is essential forventral-posterior patterning (Roel et al., 2002, Curr Biol 12,1941-1945), making LEF1 a potential candidate as the Xom-interactingprotein. It was therefore tested whether Xom interacts with LEF1 andexplored the critical domain of LEF1 involved in the interaction throughdeletion mutagenesis analysis.

It was found that very few cells co-expressed Xom and full-length LEF1;therefore. The potential interaction between Xom and LEF1 was thenexamined through an in vitro binding assay. When in vitro translation(IVT) produces of ³⁵S-labeled LEF1 or its deletion mutants were mixedwith GST-Xom or GST alone, LEF1 was pulled down by GST-Xom but not bythe control GST. The C-terminal-deleted mutant LEF1ΔHMG appeared to havemuch less affinity for Xom, whereas the N-terminal-deleted mutant ofLEF1Δ60 and LEF1Δ160 bound to GST-Xom as effectively as did thewild-type LEF1. Thus, the interaction between Xom and LEF1 appears to bedependent on the LEF1 C-terminal motif, a supposition further supportedby a LEF1/TCF transactivation assay.

To determine the potential physiological relevance of Xomtransactivation of LEF1/TCF-mediated transcription, the effect of Xom onthe expression of BMP4 downstream genes was examined, usingpromoter-luciferase analysis. The Msx2 gene is downstream of BMP4, whosepromoter contains both an LEF1/TCF binding site and a BMP4-responsiveelement (SBE) (Hussein et al., 2003, J Biol Chem 278, 48805-48814).Consistent with the potential direct involvement of LEF1/TCFs in BMP/Xomsignaling, it was found that Xom activated both the wild-type Msx2promoter and the SBE-mutated Msx2 promoter that retain only functionalLEF1/TCF binding sites.

Xom transactivates LEF1/TCF-mediated transcription is not mediatedthrough β-catenin accumulation—Previous studies have shown thatLEF1/TCF-mediated transcription is activated by β-catenin (Behrens etal., 1996, Nature 382, 638-642; Huber et al., 1996, Mech Dev 59, 3-10;Molenaar et al., 1996, Cell 86, 391-399). To explore the mechanism ofXom activation of LEF1/TCF-mediated transcription and to rule out thepossibility that Xom transactivation of LEF1/TCF-mediated transcriptionis through an increase in β-catenin protein level or nucleartranslocation, the effect of Xom expression on the protein level andintracellular distribution of β-catenin was examined in HCT116 cells.

It was found that, whereas Xom expression activates LEF1/TCF-mediatedtranscription in a concentration-dependent manner, there is nocorresponding increase (rather a small decrease) in the level of totalintracellular β-catenin protein. In addition, when the totalintracellular proteins were fractionated into the cytoplasmic andnuclear portions, no significant nuclear shifts of β-catenin wereobserved upon Xom expression (again, rather a small decrease). It wasalso found that, the domain of LEF1 that interacts with Xom locates atthe C-terminal region of the LEF1, which is separated from theN-terminal domain involved in interaction with β-catenin. It was foundthat the LEF1Δ60, a dominant negative LEF1 mutant that blocks β-catenintransactivation of LEF1/TCF-mediated transcription, promotes Xomtransactivation of the LEF1/TCF-mediated transcription in TOPFlashassay. This is consistent with the possibilities that Xomtransactivation of LEF1/TCF-mediated transcription through directinteraction.

Xom transactivates LEF1/TCFs through its N-terminal TAD—The interactionbetween Xom and LEF1 allowed us to further explored the molecularmechanism of Xom transactivation of LEF1/TCF-mediated transcription.Previous studies have shown that β-catenin binds to TCFs through itscentral Armadillo repeats but activates TCF through its C-terminal motif(van de Wetering et al., 1997, Cell 88, 789-799 and Orsulic et al.,1996, J Cell Biol 134, 1283-1300). Therefore, after identifying thecritical domain of Xom involved in LEF1/TCF interaction, the functionaldomain of Xom involved in LEF1/TCF transactivation it was furtheranalyzed. When cDNA encoding Xom or its deletion mutants wasco-transfected with the reporter construct of TOPflash into 293T cells,expression of both Xom and its C-terminal deletion mutant XomCD85activated the TOPflash almost 10-fold more than did the control cellstransfected with TOPflash only. In comparison, the ability to activatethe LEF1/TCF-mediated transcription was significantly diminished in theN-terminal-deleted mutants of Xom, the XomND55 and XomND175. TheXomND175 in particular retains little if any ability to transactivatethe LEF1/TCF-mediated transcription. Consistent with a model of Xomtransactivates LEF1/TCF-mediated transcription through directinteraction, it was found that Xom mutant that lack theLEF1/TCF-interaction motif failed to activate LEF1/TCF-mediatedtranscription during early embryogenesis. Xom is a known transcriptionalrepressor of dorsal gene expression. To determine whether the dualtranscriptional function of Xom is independent of each other, the effectof these Xom deletion mutants on dorsal gene expression was furtherexamined. When mRNA encoding Xom and its deletion mutants wereco-injected with mRNA encoding Activin and Gsc-promoter-luciferaseconstruct into the embryos at the two-cell stage, both theN-terminal-deleted mutants and the C-terminal-deleted mutants of Xomstrongly inhibited Activin-induced activation of the Gsc-promoter. Thesedata indicated that the function of Xom transactivation domain isindependent of the Xom repressor function, and that the Xomtransactivation domain (TAD) resides in its N-terminal region, which wasfurther supported with TOPflash assay during early embryogenesis.

Xom N-terminal TAD is required for ventral signaling—Xom is emerging asan essential cell fate determination factor during mesodermdifferentiation to promote the expression of ventral specific genes(Koide et al., 2005, Proc Natl Acad Sci USA 102, 4943-4948). It has beenproposed that Xom forms a positive re-enforcement loop with BMP4 topromote ventral cell fate (Schmidt et al., 1996, Development 122,1711-1721). To determine whether the Xom N-terminal TAD plays a role inXom transactivation of ventral specific genes, the effect of XomND175 ontransactivation of the BMP4 promoter during early embryogenesis wasexamined (the BMP4-luciferase construct was a generous gift of Dr. J.Feng (Zhang et al., 2002, Biochem Biophys Res Commun 293, 1412-1419 andOgawa et al., 2006, J Biol Chem 281, 18363-18369). It was found that, incontrast to Xom, XomND175 failed to transactivate the BMP4 promoter.Moreover, in comparison with the luciferase activities in controlembryos, embryos injected with XomND175 revealed less luciferaseactivities, suggesting that expression of XomND175 exerts adominant-negative effect to block the activity of endogenous Xom. Tofurther determine the effect of Xom TAD on the expression of downstreamgenes, each of the two blastomeres of embryos at the two-cell stage wasinjected with mRNA encoding Xom or XomND175. The embryos were allowed todevelop until the gastrula stage (stage 10.5) and total mRNAs wereextracted from the injected embryos and non-injected control embryos.The levels of BMP4 and Xom expression were determined by Q-PCR, usingHistone-4 as an internal control. Consistent with its role intransactivation of ventral gene expression, expression of wild-type Xomincreased the expression of BMP4. In contrast, expression of XomND175failed to enhance the expression of BMP4, suggesting that the Xom TAD isrequired for its transactivation of BMP4 expression. Moreover,consistent with the disruption of the positive auto feedback loop ofBMP4 and Xom, expression of XomND175 exerted a strong inhibitory effecton the expression of Xom itself. Together with the notion that LEF1/TCFbinding sites on the promoters of ventral specific genes are requiredfor their responsiveness to BMP4 signaling and that loss of function ofLEF1 leads to ventral defects (Roel et al., 2002, Curr Biol 12,1941-1945 and Karaulanov et al., 2004, Embo J 23, 844-856), our findingssuggest that Xom trans-activation of LEF1/TCF-mediated transcription islikely to play a critical role in ventral signaling and ventral cellfate determination.

Xom transactivation of LEF1/TCFs is essential for gastrulation—Ourbiochemical and mutagenesis studies revealed that Xom possesses aLEF1/TCF-transactivation domain in its N-terminal region. It wasidentified that a Xom mutant, XomND175 possessed negligibleLEF1/TCF-transactivation ability but retains transcriptional repressorfunction. To determine the potential physiological function of Xomtransactivation of LEF1/TCF-mediated transcription during earlyembryogenesis, the loss-of-function approach was used to explore theeffect of XomND175 expression on embryogenesis. The mRNAs encoding Xom,XomND175, and other Xom deletion mutants were injected into one of thetwo ventral blastomeres at the four-cell stage as indicated. It wasobserved that embryos injected with mRNA of XomND175 but not other Xommutants showed catastrophic effects during gastrulation. In addition tothe mutant specificity phenotype of XomND175, the XomND175 effectsappeared to be stage-specific. Unlike XomND175-m-RNA, XomND175-cDNAcaused little, if any, abnormalities during gastrulation. Closerinspection showed that the embryos injected with XomND175 mRNAprogressed through the cleavage and pregastrulation stages; however, asgastrulation begins, many large whitish gray-colored cells appear in theinjected side. As gastrulation proceeds, these cells gradually lose adefined cellular appearance, giving the embryos a “rotten” appearance.To define the cellular and histological changes associated with XomND175expression, the XomND175-mRNA-injected and control embryos was fixed,sliced them into 10-μM thin sections, and stained them with H&E andDAPI. Little histological change was associated with ND175 expression atthe cleavage and pregastrulation stages, but many large rounded-upstructures were seen at the gastrulation stage, which appeared to bedead cells. None of these large rounded-up structures in ND175-injectedembryos stained with DAPI, suggesting that they had lost normal cellularstructures, such as the nucleus. The embryos appeared to be verysensitive to the effect of XomND175. The 50% effective penetration ratewas achieved with less than 50 pg of XomND175 mRNA (data not shown). Thefinding that expression of a dominant-negative Xom mutant that lacks TADled to embryonic arrest at the gastrulation stage suggests that Xomtransactivation of LEF1/TCF-mediated transcription is essential forgastrulation. Similar findings have been noted for other BMP4 relatedtranscriptional factors, such as Bix3 (Trindade et al., 2003,Development 130, 4611-4622)

Example 2

To explore the intracellular interaction of Xom and LEF1 and itspotential cellular effect, the intracellular distribution and effect ofXom in the presence or absence of LEF1 in Hela cells was studied bystandard methods. It was found that the wild-type Xom concentrates innuclei in transfected Hela cells. However, when Xom and LEF1 wereco-transfected into Hela cells, very few cells expressed both proteins.The few cells that co-express Xom and LEF1 frequently contain giant,multi-lobular nuclei, indicating a growth-arrest effect of co-expressionof Xom and LEF1.

To prove that the growth inhibition effect of Xom is mediated throughfunctional interaction with LEF1, Xom was co-transfected with dominantnegative LEF1 (LEF1ΔHMG) into Hela cells. It was found that manytransfected cells co-expressed both Xom and LEF1ΔHMG, indicating thatthe growth-arrest effect of Xom is mediated through functionalinteraction with LEF1.

This hypothesis was further tested using a GFP and MTA cell-viabilityassay. Briefly, 5×104 cells were plated onto a 96-well cell cultureplate, 24 hours after plating, cells were transfected with 0.3 μl ofTransIT-1 and 0.1 μg of plasmid DNA. 36 hours after transfection, cellviability was measured with CellTiter 96 Aqueous No-radioactive CellProliferation Assay kit (Promega). Each transfection was repeated fourtimes to reduce the experimental variation. The results indicated thatco-expression of Xom and LEF1 significantly reduced the viability oftransfected Hela cells. Consistent with the idea that a functionalinteraction between Xom and LEF1 is important for Xom effect on cellfate, the growth-arrest phenotype was attenuated in cells co-expressingXom and LEF1 (ΔHMG).

The level of activated caspase 3 in cells transfected with Xom and LEF1or LEF1ΔHMG was examined to explore whether the cellular growth-arresteffect of the interaction of Xom and LEF1 relates to caspase activation.It was found that expression of Xom and Xom/LEF increased the levels ofactivated caspase 3, an effect that can be blocked by the apoptosisinhibitor ZVAD. The effect of Xom on caspase 3 activation was blocked byco-expression of LEFΔHMG, suggesting that a functional interactionbetween Xom and LEF1 is required for the activation of apoptosis. Thepro-apoptotic activation of LEF1 by Xom appears to be opposite theanti-apoptotic role of LEF1/TCF activation by β-catenin.

BMP4/Xom is key regulators of dorsoventral patterning. Although previousinvestigations define the role of LEF1/TCFs in dorsal development, theinteraction between LEF1/TCF and Xom suggests that LEF1/TCF plays a rolein mediating the signaling of the BMP4/Xom pathway duringventralization. To test this hypothesis, it was explored whether LEF1potentiate the ventralizing effect of Xom. When a low level of Xom orLEF1 was expressed in the dorsal blastomeres, little discernableventralizing effect was detected. However, co-expression of the Xom andLEF1 at the same concentration leads to significant dorsal inhibition.Consistent with these findings, it was found that the expression of LEF1alone results in mild inhibition of dorsal development in adosage-dependent manner. To determine the specificity of the function ofLEF1 in mediating Xom signaling, the effect of the expression ofdominant negative LEF1 (ΔHMG) on the ventralizing effect of Xom wasexplored. It was found that expression of ΔHMG rescued the ventralizedphenotype resulted from overexpression of high level of Xom, whileexpression of ΔHMG alone at the dorsal blastomeres induced littlediscernable changes.

The above results indicate that the LEF1/TCF family of transcriptionalfactors, previously known as the mediator of the Wnt/β-catenin pathway,is also the executor of the BMP4/Xom pathway. Xom and β-catenin,therefore, may compete for LEF1/TCFs for their distinguished functionsin cell fate determination. Previous studies have defined the role ofLEF1/TCFs in anti-apoptosis and dorsal development in association withβ-catenin of the Wnt pathway. Our current investigation indicates thatassociation of LEF1/TCFs with Xom may lead to cell-growth arrest andventral development. LEF1/TCFs therefore functions as the convergingpoint of Xom/BMP4 and β-catenin/Wnt signaling to mediate their combinedsignaling effect on cell fate. Beyond competition for the same family oftranscriptional factors for different phenotype and distinguished fromSmads, it was found that Xom expression reduces β-catenin protein leveland blocks β-catenin transactivation of LEF1/TCFs-mediatedtranscription. Given the implication of β-catenin transactivation ofLEF1/TCFs in patterning formation and malignant transformation of avariety cancers, further investigation of the molecular mechanismunderlying LEF1/TCFs-mediated cell fate determination of BMP/Xom areimportant for understanding the basic mechanism of embryogenesis andcarry broad therapeutic implications in neogenesis and degenerativediseases.

During our investigation, it was discovered that new component of theBMP4 pathway is critical for cell-fate determination. On the basis ofour discovery cited above, it was further discovered that BMP4 pathwayutilizes the TCF/LEF1 transcriptional factors to transduce its signal.It was found that when combined together, expression of the LEF1/TCFfactors and Xom (a component of the BMP4 pathway) induces cell death incolon cancer cells, with the efficacy of near 100% (Also effective incervical cancer cells, prostate cancer cells)-gene therapy methods.

A human homologue of Xom, Hom, (previously known as the VentX2), wascloned by a standard method and subjected to the above assays. It wasfound that Horn functions in a similar way to the Xenopus Xom.

Xom stability is critical for cell-fate determination. While expressionof wild-type Xom alone causes growth arrest in 30% colon cancer cells,expression of stable Xom causes growth arrest in 60% colon cancer cells.

The protein level of Xom is controlled by proteolysis, and is controlledby phosphorylation of Ser140/144. Methods and materials (e.g., specificphospho-antibodies) were developed to monitoring the Xom kinaseactivity, which can be used to screen for drugs effective for cancerprevention and treatment.

Example 3

As mentioned above, Hom is human homologue of the vertebrate Xom.Biochemical studies showed that Hom binds to theLEF1/TCF-transcriptional factors and blocks LEF1/TCF transactivation bythe oncogenic signaling of Wnt/β-catenin pathway. Hom locates onChromosome 10q26, a region that is frequently deleted in metastaticcancers, such as metastatic colon cancer, suggesting a role of Hom incancer formation and metastasis. To explore a potential role of Hom oncancer cell growth, Hom was transiently expressed in colon and lungcancer cells (HCT116 and H460 respectively) and the morphologicalchanges associated with Hom expression studied.

Plasmids (0.5 to 2 μg) encoding GFP-Hom or GFP alone were transfectedinto HCT116 or H460 cells. At 36 hours post-transfection, the cells werefixed with paraformaldehyde. The morphological changes associated withGFP-Hom or GFP expression were examined with phase contrast orfluorescent microscope. It was found that expression of GFP-Hom caused arounding-up phenotype in more than 80% of the colon and lung cancercells that transiently expressing GFP-Xom. In contrast, less than 10%cells expressing GFP became rounding-up in this assay.

To further define the morphological changes associated with expressionof GFP-Hom and determine whether the rounding-up phenotype is associatedwith cell growth arrest/death, TUNEL assay was performed on cancer cellstransiently expressing GFP-Hom. HCT116 or H460 cells were transientlytransfected with plasmid encoding GFP (vector) of GFP-Hom. Twenty-fourhours post-transfection, the cells were collected and stained withHoechst 33258 to reveal the nuclei. Cells with characteristic condensedchromatin and fragmented nuclei were scored as apoptotic cells. Thepercentage of cells that revealed apoptotic phenotype was determined bycounting the apoptotic cells and cells with normal morphology. At least400 cells were counted for each sample by random field selection. It wasfound that about 30% of HCT116 and H460 cells that transientlyexpressing GFP-Hom showed apoptotic figures, while about 5% cells in thecontrol GFP group revealed apoptotic figure. This result suggested thatexpression of Hom induces cancer cell death.

The cell growth arrest effects of Hom on cancer cells were alsoconfirmed by in vitro colony formation assay. It was found thatexpression of GFP-Hom lead to 100% reduction in colony formation incolon cancer cells and lung cancer cells, as compared with control cellsthat expressed GFP.

The p53 is a critical cellular factor involves in tumor suppression. Ithas been shown that p53 co-operate with other tumor suppressor gene,such as the ARF to suppress tumor cell growth. Loss of functionmutations of p53 is regarded as a major step during malignanttransformation. To determine whether p53 may play a role in Homsuppression cancer cell growth, the effects of Hom in colon in which thep53 has been deleted or mutated were examined, using HCT116 p53−/− andH1299 respectively following the experimental procedures describedabove.

It was found that expression of GFP-Hom caused apoptotic figures inabout 30% p53−/− colon and lung cancer cells, a figure similar to thecolon and lung cancer cells that had wild-type p53. The results suggestthat Hom triggered cell death in p53 independent manner. Thus, Hom mayplay a major tumor suppressor function in a large variety of cancercells, in which the p53 is mutated.

The effect of Hom on colon cancer cell growth was examined in athymicnude mice to determine whether Hom may suppress tumor formation in vivo.Nude mice were purchased from Charles' River Laboratory. HCT116 cellswas transiently transfected with a plasmid encoding GFP-Hom (GFP wasused as an indicator of successful transfection). A control HCT116 wasprepared by transfection with empty GFP-vector. For subcutaneousinjection, 5×10⁶ cells were prepared for each injection site. The cellswere released from culture dished with trypsin, which was inactivatedafterwards with complete serum. The cells was then collected with gentlecentrifugation at 400 g for 5 minutes and re-suspended with PBS to afinal concentration of 5×10⁷ cells/ml.

Two groups of tests were performed. In group 1, after transfection, theGFP-Hom or GFP transfected HCT116 cells were sorted from thenon-transfected cells, using GFP as a marker for the sorting. In group2, the transfected cells were used directly for injection without beingsorted for GFP expression (with an estimated transfection efficiency ofabout 60%). A 1-cc syringe was used to inject the cells subcutaneouslyinto nude mice at the shoulder level on the back after localsterilization with alcohol. For each site, 100 μl of cells, whichcontains 5×10⁶ cells, were injected. The injected mice were ear taggedand observed daily for up to 21 days (It usually takes 7 to 10 days fortumors to develop in nude mice). After the morphological observation,the animals were sacrificed and the tumors dissected out. The cellularand structure abnormalities of the tumors was determined with HEstaining and microscopic examination.

It was found that no tumors formed in nude mice injected with the sortedGFP-Hom-expressing HCT116 cells. In contrast, tumors were found in thecontrol nude mice injected with HCT116 cells that were transfected withGFP-vector alone. Similarly, nude mice injected with the unsortedGFP-Hom-expressing HCT116 cells had tumors, but had about 60% reductionin tumor size as compared with the control nude mice, which wereinjected with HCT116 transfected with GFP only. These results supportthat Hom inhibited tumorigensis.

To determine whether Hom expression affects cell proliferation andsurvival in vivo, the tumor samples were sectioned and subjected toTUNEL assays, where BrdU triphosphate may be incorporated into the3′-hydroxyl ends of DNA fragments (a hallmark of aopotosis) by theterminal deoxynucleotidyl transferase (TdT). The BrdU incorporation canbe detected by fluorescently labeled anti-BrdU antibodies. The TUNELassays showed active apoptosis in tumor cells expressing the GFP-Hom,while little apoptosis was detected in the tumor cells that expressingGFP only.

Other Embodiments

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A method for treating a cellular proliferative disorder in a subject,the method comprising administering to a subject in need thereof aneffective amount of a polypeptide containing SEQ ID NO: 1 or 3, whereinthe cellular proliferative disorder is a hematopoietic malignancy, or acancer that is selected from the group consisting of cervical cancer,colon cancer, lung cancer, and prostate cancer.
 2. The method of claim1, wherein the cellular proliferative disorder is a conditioncharacterized by aberrant activation of LEF1/TCF-mediated transcription.3. The method of claim 2, wherein the polypeptide lacks an LEF1/TCFtransactivation domain.
 4. The method of claim 1, wherein the cancer ischaracterized by a mutation in the p53 gene.
 5. A method for treating acellular proliferative disorder in a subject, the method comprisingadministering to a subject in need thereof an effective amount of apolypeptide comprising a fragment of SEQ ID NO: 7, wherein said fragmentof SEQ ID NO: 7 has an amino acid substitution at Ser 140 or Ser 144,and wherein said fragment contains SEQ ID NO: 3, wherein the cellularproliferative disorder is hematopoietic malignancy, or a cancer that isselected from the group consisting of cervical cancer, colon cancer,lung cancer, and prostate cancer.
 6. The method of claim 5, wherein Ser140 or Ser 144 is replaced with alanine.
 7. The method of claim 5,wherein the polypeptide has the ability to bind to LEF1/TCF and inhibitLEF1/TCF-dependent transcription.
 8. The method of claim 1, wherein thepolypeptide has the sequence of SEQ ID NO:
 1. 9. The method of claim 8,wherein the polypeptide consists of the sequence of SEQ ID NO:
 1. 10.The method of claim 1, wherein the polypeptide has the sequence of SEQID NO:
 3. 11. The method of claim 10, wherein the polypeptide consistsof the sequence of SEQ ID NO: 3.